characterizing novel interactions of transcriptional ... novel interactions of transcriptional...

Characterizing Novel Interactions of Transcriptional Repressor Proteins BCL6 & BCL6B

by

Geoffrey Graham Lundell-Smith

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Biochemistry University of Toronto

© Copyright by Geoffrey Lundell-Smith, 2017

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Characterizing Novel Interactions of Transcriptional Repression

Proteins BCL6 and BCL6B

Geoffrey Graham Lundell-Smith

Masters of Science

Department of Biochemistry

University of Toronto

2016

Abstract

B-cell Lymphoma 6 (BCL6) and its close homolog BCL6B encode proteins that are members of

the BTB-Zinc Finger family of transcription factors. BCL6 plays an important role in regulating

the differentiation and proliferation of B-cells during the adaptive immune response, and is also

involved in T cell development and inflammation. BCL6 acts by repressing genes involved in

DNA damage response during the affinity maturation of immunoglobulins, and the mis-

expression of BCL6 can lead to diffuse large B-cell lymphoma. Although BCL6B shares high

sequence similarity with BCL6, the functions of BCL6B are not well-characterized. I used

BioID, an in vivo proximity-dependent labeling method, to identify novel BCL6 and BCL6B

protein interactors and validated a number of these interactions with co-purification experiments.

I also examined the evolutionary relationship between BCL6 and BCL6B and identified

conserved residues in an important interaction interface that mediates corepressor binding and

gene repression.

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Acknowledgments

Thank you to my supervisor, Gil Privé for his mentorship, guidance, and advice, and for giving

me the opportunity to work in his lab.

Thanks to my committee members, Dr. John Rubinstein and Dr. Jeff Lee for their ideas,

thoughts, and feedback during my Masters. Thank you to Dr. Etienne Coyaud for performing the

mass spec experiments and providing help with data analysis, advice, and troubleshooting.

An incredible amount of thanks to Neil Pomroy without whom much of this work would have

been so much more difficult, if not nigh-impossible. Thanks also to Dr. Doug Kuntz, Dr. Wesley

Errington, and Dr. Yong Wei, your guidance and advice has been invaluable. A big thank you to

my fellow grad students in the lab, Andrew, Alan, Darren, Xiong, Yaseen, and the new guy, for

the advice, support, thoughtful discussion, and allowing me free reign over the lab’s music

playlist. I’d also like to thank the staff of the Department of Biochemistry, especially Carrie

Harber, who works administrative wonders.

Special thanks to my friends, you guys helped me through all the ups and downs, and all of the

craziness. T follicular helper cells, right?

Most importantly, to my mother and father, my sister Kierstin and my brother Connor, your

support has meant the world to me. Thank you so much for being there.

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Table of Contents

Contents

Acknowledgments .......................................................................................................................... iii

Table of Contents ........................................................................................................................... iv

List of Abbreviations .................................................................................................................... vii

List of Tables ................................................................................................................................. ix

List of Figures ................................................................................................................................. x

List of Appendices ........................................................................................................................ xii

1 Introduction ................................................................................................................................ 1

1.1 BTB Domains ..................................................................................................................... 1

1.2 BTB-Zinc finger proteins .................................................................................................... 1

1.3 General Mechanism of Transcriptional Repression ............................................................ 4

1.4 BCL6 ................................................................................................................................... 6

1.4.1 Introduction ............................................................................................................. 6

1.4.2 BCL6 Biological Roles ........................................................................................... 7

1.4.3 BCL6 and the Germinal Centre Reaction ............................................................... 8

1.4.4 BCL6 as Master Regulator of the Germinal Centre Reaction .............................. 10

1.4.5 BCL6 Regulation and Lymphomagenesis ............................................................ 11

1.5 BCL6B is a paralog of BCL6 ............................................................................................ 12

1.6 The Structure of the BCL6 BTB domain .......................................................................... 14

1.7 The BCL6 Interactome ..................................................................................................... 20

1.8 Protein-Protein Interactions .............................................................................................. 22

1.9 BioID ................................................................................................................................. 24

1.10 Thesis Objectives .............................................................................................................. 27

2 Materials and Methods ............................................................................................................. 28

v

2.1 Cloning and Generation of Constructs .............................................................................. 28

2.2 SDS-PAGE and Western Blots ......................................................................................... 28

2.3 Mammalian Tissue Culture ............................................................................................... 29

2.4 Analysis of BioID Results ................................................................................................ 30

2.5 Transient Transfection and Co-Immunoprecipitation ....................................................... 30

2.6 Expression and Purification of BCL6BBTB ....................................................................... 31

2.7 Circular Dichroism of BCL6B BTB in Sarcosyl .............................................................. 32

2.8 Microscale Thermophoresis .............................................................................................. 32

2.9 Crystallization of BCL6BBTB ............................................................................................ 33

2.10 Phylogenetic Analysis and Sequence Conservation ......................................................... 33

3 Results ...................................................................................................................................... 35

3.1 BioID identifies known and novel interactors of BCL6 and BCL6B ............................... 35

3.2 Validation of Identified Interactions ................................................................................. 45

3.3 Purification of the BCL6B BTB domain .......................................................................... 46

3.3.1 Constructs and Purification Strategy .................................................................... 46

3.3.2 Evaluation of stability, solubility, and folding of BCL6BBTB in the presence of

Sarcosyl ................................................................................................................. 49

3.3.3 Crystallization Trials ............................................................................................. 51

3.4 Biophysical Characterization of the interaction between BCL6B and SMRT ................. 52

3.5 Bioinformatic Analyses .................................................................................................... 55

3.5.1 Phylogenetic analysis of BCL6/B ......................................................................... 55

3.5.2 Conservation of Corepressor BBDs ...................................................................... 60

3.5.3 Conservation of the BCL6 and BCL6B lateral grooves ....................................... 60

3.5.4 Co-evolving Residues in the BTB Domain .......................................................... 64

4 Discussion ................................................................................................................................ 67

4.1 Experimental Rationale ..................................................................................................... 67

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4.1.1 Choice of Technique ............................................................................................. 67

4.1.2 Identification of Statistically Significant Interactors ............................................ 70

4.1.3 Validation of Target Interactions: ......................................................................... 70

4.2 Interactions Identified by BioID ....................................................................................... 71

4.2.1 The BCL6 BTB domain interacts with TLE1 and TLE3 ...................................... 75

4.2.2 The BCL6 BTB domain was not observed to interact with NFIA ....................... 76

4.3 BCL6B Interacts with the SMRT BBD ............................................................................ 76

4.4 Evolutionary Conservation of BCL6/B and the BBD ....................................................... 78

4.5 Co-evolution of BCL6 and BCL6B Dimerization Residues ............................................. 80

4.6 Expression and Purification of the BCL6B BTB domain ................................................. 81

5 Conclusion and Future Directions ............................................................................................ 83

5.1 Conclusions ....................................................................................................................... 83

5.2 Further Characterization of BCL6-TLE interaction ......................................................... 84

5.3 Mapping of Interactions with Repression Domain Mutants ............................................. 85

5.4 Structural Characterization of BCL6B BTB Domain and Interaction with SMRT .......... 85

References ..................................................................................................................................... 86

Appendices .................................................................................................................................. 102

vii

List of Abbreviations

AIDA: Activation Induced Cytosine Deaminase

AP-MS: Affinity Purification-Mass Spectrometry

BBD: BTB-binding Domain

BCL6: B-cell Lymphoma 6

BCL6B: B-cell Lymphoma 6 member B

BCOR: BCL6 Corepressor

BMe: Beta-mercaptoethanol

BTB: Broad, Tramtrack, Bric-a-brac

CD: Circular Dichroism

CSR: Class Switch Recombination

CRAPome: Contaminant Repository for Affinity Purification

CTF: CCAAT box-binding Transcription Factor

DLBCL: Diffuse Large B-cell Lymphoma

DNA: Deoxyribonucleic Acid

GC: Germinal Centre

HAT: Histone Acetyltransferase

HDAC: Histone Deacetylase

HSP90: Heat Shock Protein 90

KD: Dissociation Constant

MATH: Meprin and TRAF-C Homology

MST: Microscale Thermophoresis

NCOR1: Nuclear Corepressor 1

NFI: Nuclear Factor I

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Ni-NTA: Nickel-Nitrilotriacetic Acid

PCR: Polymerase Chain Reaction

PDB: Protein Data Bank

PLZF: Promyelocytic Leukemia Zinc Finger protein

PRC1: Polycomb Repressive Complex

POZ: Pox virus and Zinc Finger

PVS: Protein Variability Server

RD2: Repression Domain 2

RNA: Ribonucleic Acid

SAINT: Significane Analysis of Interactome

SEC: Size Exclusion Chromatography

SHM: Somatic Hypermutation

SMRT: Silencing Mediator of Retinoid and Thyroid receptor

STRING: Search Tool for the Retrieval of Interacting Genes/Proteins

SUMO: Small Ubiquitin-Related Modifier

TFH: T-Follicular Helper cell

Thx: Thioredoxin

TLE: Transducin-like Enhancer of split

ZF: Zinc Finger

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List of Tables

Table 1: Significant interactors for BCL6 identified by BioID. ................................................... 36

Table 2: Significant interactors for BCL6B identified by BioID ................................................. 38

Table 3: Summary of BCL6/B homologs identified in representative organisms ........................ 56

Table 4: Summary of NCOR1, SMRT, and BCOR homologs identified in representative

organisms. ..................................................................................................................................... 62

x

List of Figures

Figure 1.1: Distance tree of human BTB-ZF proteins .................................................................... 2

Figure 1.2: Schematic of a generic BTB-ZF protein ...................................................................... 2

Figure 1.3: Structures of the BTB domain dimer ........................................................................... 4

Figure 1.4: Schematic of the BCL6 protein .................................................................................... 7

Figure 1.5: Overview of the Germinal Centre Reaction ................................................................. 9

Figure 1.6: Summary of genes suppressed by BCL6 during the Germinal Centre Reaction ....... 11

Figure 1.7: Comparison of BCL6 and BCL6B ............................................................................. 13

Figure 1.8: Crystal structure of the BCL6 BTB domain ............................................................... 15

Figure 1.9: Secondary structure topology of the BTB domain homodimer ................................. 16

Figure 1.10: Crystal Structures of the BBD peptide interaction with BCL6 ................................ 17

Figure 1.11: Illustration of the NCOR/SMRT complex bound to BCL6 ..................................... 19

Figure 1.12: The BCOR PRC1-like core complex ....................................................................... 19

Figure 1.13: STRING Network of known BCL6 Interactors ....................................................... 21

Figure 1.14: STRING Network of known BCL6B interactors ..................................................... 22

Figure 1.15: Method of BioID protein identification .................................................................... 26

Figure 3.1: STRING Network of Interactors identified in BioID experiments ............................ 44

Figure 3.2: Western Blot of Inputs and BCL6 interaction validation by CoIP ............................. 46

Figure 3.3: SDS-PAGE gel showing the purification of Thx-BCL6BBTB by Nickel Affinity

Chromatography ........................................................................................................................... 47

Figure 3.4: Gel filtration profile of Thioredoxin-BCL6BBTB........................................................ 48

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Figure 3.5: Structure of the sarcosyl anionic detergent molecule ................................................. 48

Figure 3.6: SDS-PAGE gel showing the purification of BCL6BBTB following cleavage of 6xHis-

SUMO tag ..................................................................................................................................... 49

Figure 3.7: Circular Dichroism spectra of BCL6B protein. .......................................................... 50

Figure 3.8: Temperature profile of BCL6B. ................................................................................. 51

Figure 3.9: Gel filtration profile of BCL6BBTB run on an se70 analytical column ....................... 52

Figure 3.10: SMRT-BBD-BTB domain interaction binding curves ............................................. 53

Figure 3.11: Phylogenetic representation of query species and presence of BCL6/B homologs. 57

Figure 3.12: Phylogenetic Tree of BTB domains ......................................................................... 59

Figure 3.13: Graphical representations of the sequence alignments of BBDs from NCOR1,

SMRT, and BCOR ........................................................................................................................ 61

Figure 3.14: Shannon sequence entropy mapped to the surface of the BCL6 and BCL6B BTB

domain monomers ......................................................................................................................... 63

Figure 3.15: Contact map of co-varying BTB residues ................................................................ 65

Figure 3.16: Sequence alignment of the BCL6 and BCL6B BTB domains with co-evolving inter-

dimer contact residues highlighted and connected to their “co-evolving” partner residue by

arcing lines .................................................................................................................................... 65

Figure 3.17: Location of co-evolving residues in the BCL6 BTB homodimer. ........................... 66

Figure 4.1: Modes of BCL6B-mediated repression of target genes ............................................. 74

Figure 4.2: Schematic of conserved TLE1/3 domains .................................................................. 75

xii

List of Appendices

Table A1: BCL6/B Proteins included in bioinformatic analyses ................................................ 102

Figure A1: Unrooted phylogenetic tree of the BTB domains. .................................................... 103

Figure A2: Unrooted phylogenetic tree of the full-length BCL6/B proteins .............................. 104

Figure A3: Unrooted phylogenetic tree of the ZF regions .......................................................... 105

Table A2: Summary of NCOR1/SMRT repressor complex members and PRC-like complex

members identified in representative organisms ......................................................................... 106

1

1 Introduction

1.1 BTB Domains

The BTB Domain is a conserved structural fold found in proteins from a wide variety of

organisms including viruses and most eukaryotes. It was first identified in three different

Drosophila transcription factors, Broad Complex, Tramtrak, and Bric-à-brac, from which it takes

its name (Zollman et al., 1994). The BTB domain is also known as the POZ domain in scientific

literature, owing to the discovery of homology between poxvirus proteins and various zinc finger

proteins (Bardwell and Treisman, 1994). The BTB domain is approximately 120 amino acids

long and usually functions as a mediator of protein-protein interactions (Stogios et al., 2005).

BTB domains share a conserved tertiary fold consisting of five alpha helices and three beta

strands. Generally, the BTB domain is present in one copy at the N-terminus of a protein, and is

often coupled with one or more different domains, notably the MATH, Kelch, Zinc Finger, and

Ion transport domains (Perez-Torrado et al., 2006). Association of the BTB domain with these

different domains is useful in classifying BTB-containing proteins into different families based

on structure and function.

1.2 BTB-Zinc finger proteins

One of the more well-characterized BTB families is the BTB-Zinc finger group of proteins, of

which 49 different members have been found in humans (Siggs and Beutler, 2012)(Figure 1.1).

All members of this family share a common architecture, with an N-terminal BTB domain

connected by an intrinsically disordered middle linker region of variable length to a set of C-

terminal C2H2/Kruppel-type Zinc finger domains that vary in number for each BTB-ZF protein

(Stogios et al., 2005)(Figure 1.2).

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Figure 1.1: Distance tree of human BTB-ZF proteins. Human BTB-ZF proteins are displayed in a distance tree.

Proteins are labelled by their ZBTB identifier and common gene name, if applicable.

Figure 1.2: Schematic of a generic BTB-ZF protein. Protein is displayed from N-terminus to C-terminus, left to

right, and consists of a BTB domain in blue, an intrinsically disordered middle linker region, and set of C-terminal

zinc fingers. Domains are annotated according to known function.

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Each domain of a BTB-ZF protein carries out a different function: the BTB domain mediates

interaction with different protein partners and helps determine biological role. The zinc fingers

bind to different regions of DNA in a specific manner to affect expression of target genes, and

may also play a role in protein-protein interaction. The middle linker region, though less well-

conserved than the other regions, also plays a role in protein-protein interaction in some proteins,

and is a location for post-translational modification of the BTB-ZF proteins (Costoya et al.,

2008; Huang et al., 2014).

BTB-ZF proteins are obligate dimers with a well-conserved dimerization interface (Stogios et al.,

2005). The crystal structures of several different BTB-ZF proteins have been solved, and despite

modest conservation of sequence identity, all BTB domain structures exhibit a similar structure

with a characteristic “butterfly” appearance (Figure 1.3). An important aspect of the BTB

domain in BTB-ZF proteins is the N-terminus, which forms an additional β-strand and α-helix

that are integral to BTB dimer formation. The dimerization interface is hydrophobic and

composes nearly one quarter of the monomer surface(Ahmad et al., 2003). Though most BTB-

ZF proteins appear to be obligate homodimers, some are able to form heterodimers with other

BTB-ZF proteins, including Nacc1 and Miz1, Miz1 and BCL6 , and possibly BCL6 and BCL6B

(Okabe et al., 1998; Stead and Wright, 2014).

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Figure 1.3: Structures of the BTB domain dimer. Six structures have been solved in our lab representing the BTB

domains from BCL6, PLZF, FAZF, Kaiso, LRF, and Miz1. Despite each domain having low shared sequence

identity (45% or less) and diverse functions, all proteins form similar dimeric structures. Monomers of each domain

forming the dimers are coloured a different shade of blue. Figure adapted from Ahmad et al., 1998, 2003; Ghetu et

al., 2008; Stogios, 2008.

In part due to the DNA-binding activity of their zinc finger domains, BTB-ZF proteins serve as

transcriptional activators and repressors. For instance, the ability of BCL6 to act as a

transcriptional repressor through interactions with the corepressors SMRT, NCOR1, and BCOR,

has been well-documented (Ahmad et al., 2003; Ghetu et al., 2009). Other BTB-ZF proteins,

such as PLZF, have also been found to recruit corepressor complexes to their target genes (Choi

et al., 2014), though whether all BTB-ZF proteins function as transcriptional repressors is not yet

known.

1.3 General Mechanism of Transcriptional Repression

In eukaryotes, controlling the initiation of transcription is the primary method of regulating gene

expression. Transcription is carried out by RNA polymerase II (Pol II), which, to initiate

transcription, requires the activity of many other proteins, called general transcription factors, to

properly position Pol II and unwind DNA at the initiation site. There are a number of means

available to eukaryotes to regulate this initiation of transcription. One mechanism concerns

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controlling the rate of initiation of transcription. The availability of basal transcription machinery

is a limiting factor on transcription initiation and different genes compete for these general

transcription factor by using sequence-specific transcription factors to recruit them (Hager et al.,

2009). Transcription factors may also activate or repress transcription by directly interacting with

cis-regulatory sequences at or near promoters. Some transcriptional repressors can physically

block activators or RNA polymerases from binding or moving along target DNA, as in the case

of certain Hox genes binding the Distal-less conserved regulatory element to repress

transcription in Drosophila (Uhl et al., 2016).

Another mechanism of regulation is the manipulation of the structure of DNA in the nucleus to

prevent access to target genes. Higher order structure of DNA is both useful and necessary for

packing DNA into the nucleus of the cell. As part of this higher order structure, DNA winds

around proteins, called histones, to form a repeating unit of protein-bound DNA. These units are

called nucleosomes. The basic format of the nucleosome consists of 146bp of DNA wound

around an octamer of histones, two apiece of H2A, H2B, H3, and H4, with a 54bp linking region

of DNA connecting one nucleosome to the next. A key feature of nucleosomes is that the

histones within tend to be compacted except at their N-termini, where a disordered region

protrudes; this disordered region is available for manipulation by different enzymes.

The structure of chromatin can be changed from compacted (and therefore inaccessible) to open

(and accessible) by covalently modifying residues on the histone tails. This is a reversible

process, allowing switching between open and condensed chromatin (called euchromatin and

heterochromatin, respectively). Some of the modifications available are acetylation, methylation,

ubiquitination, sumoylation and phosphorylation, and are proposed to form a histone code

readable by other histone-modifying enzymes (Strahl and Allis, 2000; Zhang et al., 2015).

Acetylation and methylation in particular figure prominently in the activation and inactivation of

chromatin for transcription. Acetylation is catalyzed by a group of enzymes called histone

acetyltransferases (HATs), of which there are three main families: GNAT, MYST, and

CBP/p300, identified based on sequence similarity and acetylation motifs (Sterner and Berger,

2000). Acetylation negates the positive charge of lysine residues on the histone tails and de-

condenses chromatin, opening it up to a form that enables the recruitment of transcription

factors. In general, it is the tails of H3 and H4 histones that are targeted by this activity. In

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opposition to transcription-activating acetylases are histone deacetylases (HDACs) that remove

acetyl groups from the lysines and induce chromatin to form a condensed structure. In

vertebrates 18 different HDACs have been identified and categorized into 4 different subclasses

based on structure, enzymatic function, subcellular localization, and expression patterns

(Haberland et al., 2009). Certain histone lysine methylation modifications, for instance the

methylation of lysine 27 on H3 histones, are associated with silencing of gene transcription,

maintaining the chromatin status over longer periods of time (Rose and Klose, 2014).

Methylation of histones is carried out by methyltransferases and reversed by demethylase

enzymes. The H3K27me modification in particular is carried out by the polycomb group of

transcriptional repressors (Schlesinger et al., 2007).

Some histone modifying enzymes are able to interact directly with DNA or DNA-binding

transcription factors. Others, as in the case of HDACs, require other proteins to mediate

interactions in order to direct their specific activity and be recruited to the right histone

substrates. The HDACs themselves have no intrinsic DNA binding ability; it is through forming

(sometimes very large) complexes that they are directed to their target genes. Canonically,

HDAC complexes include transcriptional repressors with DNA-binding domains, and

corepressors that serve as scaffolding proteins to bridge HDACs to the target sequences. NCOR,

SMRT, mSin3A, MTA3, and CtBP are corepressors that are known to interact with a variety of

transcriptional repressors including the BTB-ZF protein BCL6 (Ahmad et al., 2003; Jaye et al.,

2007; Mendez et al., 2008) . They serve as scaffolding proteins to recruit HDAC and

demethylase complexes for the silencing and repression of target genes (Perissi et al., 2010).

1.4 BCL6

1.4.1 Introduction

BCL6 is a 706 residue transcription factor and one of the most well-characterized members of

the BTB-ZF family. It has an N-terminal BTB domain extending from residues 5-129 that

mediates protein-protein interactions (Ahmad et al., 2003; Chang et al., 1996). This is followed

by an intrinsically disordered middle linker region that contains a conserved RD2 (repression

domain 2) region that also mediates certain repression activity and protein stability (Huang et al.,

2014; Seyfert et al., 1996). At the C-terminus, BCL6 has a set of six zinc fingers, spanning

7

residues 518-681 that bind to specific target DNA sequences (Kawamata et al., 1994) (Figure

1.4). BCL6 was first identified because of its role in the development of non-Hodgkins

lymphoma by way of chromosomal translocation at the 3q27 locus (Lo Coco et al., 1994).

Figure 1.4: Schematic of the BCL6 protein. Different domains of BCL6 are presented here. RD2: Repression

domain two; ZF: zinc finger.

1.4.2 BCL6 Biological Roles

Because of its involvement in lymphomagenesis, BCL6 has been extensively studied to elucidate

its roles in normal and oncogenic biological processes. Early on, BCL6-deficient mouse models

were created to study the effects of BCL6 loss. BCL6 -/- models displayed severe growth

retardation and ill health. Greater than 80% of these mice experienced premature death caused by

Th2 inflammatory disease, and all of these mice exhibited a failure to form germinal centres and

did not generate an immunoglobulin response to T cell-dependent antigen invasion (Dent et al.,

1997; Fukuda et al., 1997). Mouse models have also been engineered to test the constitutive

expression of BCL6. These mice exhibited increased germinal centre (GC) formation followed

by a lymphoproliferative disease which developed into lymphoma displaying many of the

hallmarks of human diffuse large B-cell lymphoma (DLBCL) (Cattoretti et al., 2005).

Additional mouse models explored disruption to the functional domains of BCL6. For the BTB

domain, mice with a BCL6BTBmutant knock-in were created. Mutations introduced to the BTB

domain disrupted a known protein-protein interaction region, called the lateral groove (Ahmad et

al., 2003; Huang et al., 2013). Unlike BCL6 knockout mice, these mice grew normally, had

normal Th2 maturation, no lethal inflammatory phenotype, and normal chemokine development

in macrophages. However, they exhibited impaired GC formation resulting in impaired

immunoglobulin affinity maturation, but had normal T-follicular helper (TFH) cell development.

(Huang et al., 2013).

BCL6 mice were also created with mutations to the conserved RD2 region in their middle linker,

which disrupted the interaction region. Similar to BTB-mutant mice, these mice had normal birth

8

and growth. They also exhibited complete loss of GC formation, and showed partial, but not full

impairment of normal TFH development. They likewise had no lethal inflammatory phenotype

and no macrophage deregulation (Huang et al., 2014).

BCL6 plays a key role in hematopoiesis and immune system development, with both the BTB

domain and RD2 domain interactions being characterized for their importance in B cell

development in the germinal centre reaction, formation and development of TFH cells, TH2 and

TH17 differentiation, and TH

2 lethal inflammatory phenotype mediated by expression of

inflammatory chemokines in macrophages and helper T cells (Hatzi and Melnick, 2014; Jardin et

al., 2007; Mathew et al., 2014; Ye et al., 1997).

1.4.3 BCL6 and the Germinal Centre Reaction

BCL6 has well-characterized roles in the humoral immune response. Specifically, BCL6 is a

master regulator of the Germinal Centre (GC) reaction. Germinal centres are transient and

dynamic cellular compartments whose formation and maintenance are tightly regulated (Allen et

al., 2007). These structures form in the secondary lymph nodes in response to T-cell dependent

antigen activation (Ranuncolo et al. 2008). They are regions for somatic hypermutation of

immunoglobulin genes, class-switch recombination, and rapid clonal expansion of antigen-

activated B-cells. The ultimate goal of this process is to generate high-affinity antibodies against

specific antigens (MacLennan, 1994).

The humoral response begins with naïve B-cells circulating in secondary lymphoid tissues where

they may encounter antigens and become antigen-engaged. These cells then migrate to the border

of follicles and T-cell zones within the lymph nodes (Okada et al., 2005). Engaged B-cells begin

to proliferate and interact with T-follicular helper cells (Kerfoot et al., 2011). These B-cells may

then differentiate to low-affinity plasma cells or, alternatively, they can enter the GC reaction,

becoming GC precursor cells. At this point they begin to upregulate BCL6 protein expression

and aggregate into GC clusters (Kitano et al., 2011).

Once early GCs are established, B Cells undergo rapid proliferation, causing massive GC

expansion. After several days of proliferation the GC becomes fully formed, and is polarized into

two different regions, the light zone and the dark zone (Allen et al., 2007). B cells within the

dark zone are called centroblasts, and undergo massive clonal expansion and somatic

9

hypermutation (SHM) (Gatto and Brink, 2010). Somatic hypermutation is the result of single and

double-strand DNA damage mediated by activation induced cytosine deaminase (AIDA)

(Muramatsu et al., 2000). In normal GC reactions, these mutations are targeted to the variable

region of immunoglobulin genes, generating mutant clones of antibodies covering a wide array

of affinities for the specific activating antigen. Following SHM, these cells migrate towards the

light zone. B cells in the light zone, termed centrocytes, no longer undergo proliferation and

instead begin the process of selection and differentiation (McHeyzer-Williams et al., 2012).

Positive selection of centrocytes expressing high affinity antibodies is mediated by TFH cells and

centrocytes expressing low-affinity immunoglobulin either undergo apoptosis or are returned to

the dark zone for further SHM (Victora, 2014). Within the light zone, AIDA also mediates class

switch recombination to produce different types of immunoglobulin (Muramatsu et al., 2000).

High affinity antibody-expressing centrocytes then undergo terminal differentiation into

antibody-secreting plasma cells or long-lived memory B cells (Victora and Nussenzweig,

2012)(Figure 1.5).

Figure 1.5: Overview of the Germinal Centre Reaction. Adaptive immune response begins with activation of

naïve B cells (green) by foreign antigens, causing them to differentiate into centroblasts (yellow), which undergo

clonal expansion in the dark zone of newly formed germinal centres. As centroblasts proliferate, somatic

10

hypermutation (lightning bolt) of their immunoglobulin genes occurs, introducing base pair changes into the variable

regions of these genes, resulting in a change of amino acid sequence in some cases. These centroblasts then migrate

to the light zone and differentiate into centrocytes (red). In the light zone, T cells (light blue) and follicular dendritic

cells (light green) help in selection for high-affinity binding to the activating antigen. Centrocytes with unfavourable

binding are either marked for apoptosis (bottom) or returned to the dark zone for more rounds of SHM (top).

Centrocytes with favourable binding then undergo terminal differentiation to either long-lived memory B cells (dark

blue) or antibody-secreting plasma B cells (purple). A subset of centrocytes undergo immunoglobulin class-switch

recombination (CSR) prior to differentiation (Klein and Dalla-Favera, 2008).

1.4.4 BCL6 as Master Regulator of the Germinal Centre Reaction

BCL6 is highly expressed in dark zone B cells, and serves as the master regulator of B cell

programming in the GC reaction (Cattoretti et al., 1995; Ci et al., 2009). The dark zone is

characterized by somatic hypermutation and clonal expansion. As such, within centroblasts

BCL6 works to facilitate tolerance to DNA damage and simultaneous rapid proliferation (Hatzi

and Melnick, 2014). To carry out these programs, BCL6 represses several different sets of genes.

For example, BCL6 represses DNA damage-sensing and checkpoint genes: DNA damage sensor

ATR, cell cycle arrest genes CHEK1 and CDKN1A, TP53 tumor suppressor, and growth arrest

and DNA-damage induced gene GADD45A (Phan and Dalla-Favera, 2004; Phan et al., 2005;

Ranuncolo et al., 2007), allowing B cells to sustain proliferation and resist DNA damage-induced

apoptosis. BCL6 also prevents premature termination of affinity maturation by repressing B cell

signalling genes such as CD69, CD44, and STAT1 (Shaffer et al., 2000) and suppresses genes

that induce terminal B-cell differentiation of centroblasts by repressing BLIMP1 (aka

PDRM1)(Shaffer et al., 2001). Additionally, BCL6 also represses several oncogenes in the GC

reaction, including BCL2, c-Myc, and cyclin D1. It has been found, however, that these

oncogenes become derepressed, even with BCL6 active, in DLBCL (Ci et al., 2009; Hatzi and

Melnick, 2014) (Figure 1.6).

11

Figure 1.6: Summary of genes suppressed by BCL6 during the Germinal Centre Reaction. Genes have been

separated with regards to the function of their products. The dotted line separates genes that act upon SHM and

proliferation from those involved in activation and differentiation of B cells. By suppressing these gene targets,

BCL6 facilitates SHM and proliferation (top), while preventing premature activation (bottom).

1.4.5 BCL6 Regulation and Lymphomagenesis

In the GC reaction, BCL6 promotes survival and proliferation, leading GC B cells to exhibit

some of the hallmarks of cancer: they proliferate rapidly, evade growth checkpoint controls, and

tolerate elevated amounts of genomic instability. The regulation of BCL6 is therefore essential to

prevent malignant transformation. BCL6 expression is regulated in GC B cells both at the

transcript and protein levels. Signalling by interleukin 21 receptor (IL21R) induces BCL6

expression at the point of B cell activation, and transcription factors IRF8 (interferon regulatory

factor 8) and MEF2B (myocyte enhancer factor 2B) upregulate transcription of the BCL6 gene

(Lee et al., 2006; Linterman et al., 2010; Ying et al., 2013). Following affinity maturation, BCL6

12

transcription is repressed primarily by IRF4, but self-repression of BCL6 also occurs (Saito et

al., 2007).

At the protein level, BCL6 is stabilized by HSP90 chaperone (Cerchietti et al., 2009). In

opposition to this, a number of post-translational modifications act to regulate BCL6 activity.

The RD2 domain is inactivated by the acetylation of key lysine residues, carried out by

EP300/CREBBP (Cerchietti et al., 2010; Huang et al., 2014). BCL6 is also targeted for

ubiquitination and proteasomal degradation by FBXO11 as part of the SCF ubiquitin ligase

complex following phosphorylation of BCL6 by mitogen-activated protein kinase (Duan et al.,

2012; Niu et al., 1998).

Dysregulated BCL6 expression is a frequent occurrence in germinal centre-derived B cell

lymphomas, as is aberrant genetic alteration to pathways regulated by BCL6. Chromosomal

translocations at the 3q27 locus upstream of BCL6 are characteristic of 40% of diffuse large B

cell lymphomas (Ohno, 2006), and somatic mutations have also been found in this regulatory

region (Jardin and Sahota, 2005). These genetic alterations interfere with the 5’ region of the first

Bcl6 intron governing the expression of BCL6, and can cause constitutive expression of the

BCL6 transcript. Additionally, oncogenes such as BCL2, c-Myc, and CCND1 normally

repressed by BCL6 in the GC reaction are found to be derepressed in DLBCL (Ci et al., 2009).

These activating mutations to BCL6 maintain GC B cells in a proliferative and pro-survival state

susceptible to oncogenic transformation.

1.5 BCL6B is a paralog of BCL6

Originally identified in 1998 by Okabe et al., BCL6B (also known as BCL6-associated zinc

finger or BAZF) is a 480 aa BTB-ZF transcription factor that shares 65% sequence identity with

BCL6 at the BTB domain and 94% sequence identity with BCL6 at the c-terminal zinc finger

region (Hartatik et al., 2001) (Figure 1.7). BCL6B’s middle linker region is considerably shorter

than that of BCL6 and the two share very little sequence homology except for a conserved 17

residue sequence, called the Repression Domain 2 (RD2), known to be associated with

repression activity in both BCL6 and BCL6B (Huang et al., 2014; Okabe et al., 1998). BCL6 and

13

BCL6B have been found to bind the same target DNA sequence, similar to the STAT6-binding

sequence (Hartatik et al., 2001).

Figure 1.7: Comparison of BCL6 and BCL6B. A) The domains and identity shared by those domains between

BCL6 (top) and BCL6B (bottom) is displayed here. RD2 domains in the middle linker region are also noted, due to

their shared conservation. B) Alignment of the BTB and ZF domains (top and bottom, respectively) of BCL6 and

BCL6B. Conserved resides are coloured blue, similar residues are coloured yellow.

There are some notable differences between BCL6 and BCL6B. Expression-wise, BCL6 is

expressed at low levels in many tissues and highly expressed in immune and hematopoietic

tissue. BCL6B also has fairly low-level ubiquitous expression (Forrest et al., 2014) and has been

found upregulated in mouse heart and lung tissues, and in premature B cells in humans, but not

in mature B cells in which BCL6 is found to be expressed in abundance. This indicates that

BCL6B may play a role in early B cell development (Sakashita et al., 2002).

BCL6B knockout mice have a milder phenotype than BCL6-/- mice. As previously stated, BCL6-

deficient mice have no germinal centre formation, the mice are severely growth retarded, and die

prematurely due to a lethal Th2 inflammation phenotype (Dent et al., 1997). BCL6 deficient

mice also have reduced hematopoietic progenitor cell production and aberrant T-cell activation.

In contrast, BCL6B deficient mice are born fertile, grow to normal size, and have normal

lifespans. BCL6B deficient mice do, however, have nearly identical hematopoietic progenitor

14

cell phenotype to BCL6-deficient mice and show similar aberrant T-cell activation and defects in

memory T-cells (Broxmeyer et al., 2007).

1.6 The Structure of the BCL6 BTB domain

The structure of the BCL6 BTB domain has been solved by x-ray crystallography to a resolution

of 1.3Å by Dr. K.F. Ahmad, a previous member of the Privé lab (Figure 1.8) (Ahmad et al.,

2003). The structure exhibits a characteristic tightly interwound butterfly shape composed of two

monomer subunits. Each monomer is composed of a central scaffolding made up of a cluster of

α-helices flanked by short β-sheets at the top and bottom of the molecule. The BTB domain has

an extensive hydrophobic dimerization interface, and the structure shows an N-terminal β-strand

is domain swapped between the two monomers. As part of the dimerization interface, the N-

terminal β1 element forms a two-stranded anti-parallel beta sheet with β5’ from the other BCL6

monomer. The principle dimer contacts between the two BTB subunits are mediated by β1, α1,

α2, β5, and α6, and 25% of the BTB monomer’s surface area is buried upon dimerization

(Figure 1.9) (Ahmad et al., 2003; Stogios et al., 2005). Nearly all of the residues that stabilize

the BCL6 BTB domain dimer are well-conserved within the BTB-ZF family.

15

Figure 1.8: Crystal structure of the BCL6 BTB domain. Each chain of the dimer is coloured differently and N-

and C-termini are labelled.

16

Figure 1.9: Secondary structure topology of the BTB domain homodimer. Secondary structure elements of each

chain are labelled. Adapted from Ahmad et al., 1998.

In addition, the interactions between BCL6 and the corepressors BCOR and SMRT have also

been characterized structurally (Figure 1.10). The minimal interaction regions of SMRT and

BCOR were identified by domain mapping and biophysical characterization. In SMRT, residues

1414-1441 form the minimal BTB-binding domain (BBD). In BCOR, the BBD consists of

residues 498-514, and there is almost no sequence similarity between the BCOR and SMRT

BBDs.

BBD fragments were co-crystallized with the BTB domain, binding in a 2:2 ratio in a shallow

groove formed at the BTB-dimer interface, known as the lateral groove. Both SMRT and BCOR

BBDs associate with the same region of the BTB dimer (Figure 1.10B). The BBD peptide forms

contacts with both subunits of the BTB dimer in an extended conformation within the lateral

groove. Mutations of residues N21 and H116 of the lateral groove of BCL6 completely abrogate

17

binding with corepressor BBDs while still maintaining the BTB homodimer fold (Ahmad et al.,

2003; Ghetu et al., 2008)

Figure 1.10: Crystal Structures of the BBD peptide interaction with BCL6. A) 2:2 complex of BCL6BTB and

either SMRT-BBD (left) or BCOR BBD (right). In both structures the BBD binds to the lateral groove of the BTB

dimer. B) Closer view of the SMRT BBD (pink) bound to the lateral groove.

The interaction between the BCL6 BTB domain and the corepressors NCOR1, BCOR, and

SMRT is vital to BCL6 function in B cell development and lymphomagenesis. Disruption of this

interaction, whether through mutation to important lateral groove residues or the use of peptides

and small molecules, results in a failure to form GCs (Huang et al., 2013; Polo et al., 2004).

Furthermore, inhibition of BCL6 interactions with these corepressors de-represses the key

checkpoint genes ATR, p53, and CDKN1A (Cerchietti et al., 2009, 2010). As part of the BCL6

18

repression program, these corepressors serve as scaffolds for the assembly of various repression

complexes that carry out their function through modification of chromatin.

NCOR1 and SMRT are large, homologous proteins of approximately 2500 residues each. SMRT

and NCOR1 can interact with HDAC3 to carry out repression by deacetylation (You et al.,

2013). However, SMRT and NCOR1 also interact with other HDACs in a context-dependent

manner. These include class II HDACs 4,5, and 7, and HDAC1 through interaction with mSin3a

(Downes et al., 2000; Fischle et al., 2002; Nagy et al., 1997). The core NCOR/SMRT complex

consists of TBL1X or its homolog TBL1XR1, GPS2, and HDAC3 (Figure 1.11); within this

complex, NCOR1 and SMRT appear to be interchangeable (Oberoi et al., 2011). The BBDs for

SMRT and NCOR1 are 70% identical and share the most conserved residues that contact the

BCL6 BTB lateral groove.

On the other hand, BCOR shares little sequence identity and recruits an entirely different

repression complex compared to SMRT/NCOR1 and mediates repression through different

chromatin modifications. BCOR is an approximately 1700 amino acid protein that recruits a

PRC1-like complex with demethylation and ubiquitination activity (Gearhart et al., 2006). The

core components of the BCOR complex are the demethylase KDM2B, PCGF1, ubiquitin E2

ligases RING1 and RNF2, RYBP, and SCF ubiquitin ligase SKP1 (Gearhart et al., 2006;

Sánchez et al., 2007) (Figure 1.12). This complex may mediate repression through

ubiquitination of histones and promote gene silencing (Farcas et al., 2012; Sánchez et al., 2007;

Wang et al., 2004).

19

Figure 1.11: Illustration of the NCOR/SMRT complex bound to BCL6. BCL6 directly recruits the corepressors

NCOR and SMRT (in bold) at enhancer or promoter regions, which then form a complex that includes HDAC3 and

other members (in blue) of the HDAC family, to repress transcription of a target gene.

Figure 1.12: The BCOR PRC1-like core complex. BCOR recruits many different proteins (cyan) as part of its

complex, which ubiquitinates histones to mediate gene silencing. BCL6 directly interacts with BCOR (bold) at

promoter regions to recruit this complex.

20

BCL6 displays the ability to bind both BCOR and NCOR1/SMRT simultaneously in a ternary

complex, interacting with one of each corepressor through its two identical lateral grooves (Hatzi

et al., 2013). BCL6 forms this ternary complex at promoter regions of target genes, and

preferentially recruits SMRT to repress genes at enhancer regions. Thus the ability to form

hybrid complexes and recruit different repression complexes enhances the functionality that

BCL6 brings to genes targeted for repression (Hatzi and Melnick, 2014).

The BTB domain is not the only region of BCL6 with repressor activity and both the BTB

domain and RD2 domain are capable of acting autonomously by recruiting distinct sets of

corepressors (Huang et al., 2013). As such, corepressor binding to the lateral groove mediates

GC repression; while the other functions of BCL6 in different cell types are mediated by distinct

mechanisms.

1.7 The BCL6 Interactome

The BioGRID interaction repository database lists 117 unique protein interactors for BCL6 and

only seven unique interactors for BCL6B(Chatr-Aryamontri et al., 2015). Among the interactors

for BCL6 are the members of the NCOR1/SMRT repression complex and associated HDACs

1,2,3,4,5, and 7 (Bereshchenko et al., 2002; David et al., 1998; Lemercier et al., 2002), BCOR

and associated members of the PRC1-like complex, and the corepressors mSin3a and RUNX1T1

(Dhordain et al., 1998; Wong and Privalsky, 1998). In addition to BCL6B, BCL6 has been found

to interact with fellow BTB-ZF proteins PLZF (Dhordain et al., 2000), Nacc1 (Korutla et al.),

and LRF (Davies et al., 1999), and the transcription factors c-Jun, JunD, and JunB (Vasanwala et

al., 2002), Sp1 (Lee et al., 2002), and BCL11A (Nakamura et al., 2000). BCL6 also directly

interacts with regulatory proteins HSP90, MAPK (Niu et al., 1998), FBX011 (Duan et al., 2012),

and is post-translationally modified by CREBBP (Huang et al., 2014). As part of its role in TFH

differentiation, BCL6 interacts with CUL3 (Mathew et al., 2014), and may interact with

corepressor protein Amino terminal Enhancer of Split (AES) (Rolland et al., 2014).

The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING—von Mering et al.,

2003) is a useful tool for analysis of protein-protein interactions in a comprehensive manner.

Important interactors for BCL6 are displayed, using STRING, in Figure 1.13. Identified

interactors for BCL6B consist of BCL6, HDAC1, and mSin3a (Takenaga et al., 2003) (Figure

1.14).

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Figure 1.13: STRING Network of known BCL6 Interactors. Interactors with BCL6 are presented as nodes

connected by edges to BCL6 and other interaction partners. The number of interactors was capped at no more than

twenty, with the top twenty interactors represented here based on the strength of evidence of their interaction with

BCL6. Proteins are coloured with similar interactors according to their functional modules, indicating these proteins

are members of the same complex or part of the same biological process. Generated from STRING database (von

Mering et al., 2003).

22

Figure 1.14: STRING Network of known BCL6B interactors. STRING representation of all identified BCL6B

interactors in the literature, including BCL6, Sin3A, and HDAC1. ARID1A and ARID1B interactors have not been

identified as interacting with BCL6B, though homologs in mice have been. Generated from STRING database (von

Mering et al., 2003).

1.8 Protein-Protein Interactions

The majority of proteins act to carry out their biological functions as part of a complex of two or

more proteins. In order to elucidate the roles that a protein plays, a first step is to identify the

interactions that protein takes part in. These protein-protein interactions can be thought of as

permanent or transient, and strong or weak. A large number of multi-protein complexes are made

up of protein-protein interactions existing on this spectrum from stable to transient.

Supramolecular assemblies and molecular machines may form more robust interactions, while

23

important biological processes involving immunity, metabolism, signal transduction, and gene

expression more often make use of weak, transient interaction with weaker binding affinities

(Robinson et al., 2007).

Strong interactions, often stabilized by one or more effector molecules, are maintained for longer

periods of time and have KD constants in the nanomolar range. In comparison, weak transient

interactions exist for much shorter times and have KD values in the micromolar range (Nooren

and Thornton, 2003). Stable interaction interfaces tend to be large, and contain a greater number

of hydrophobic residues than the rest of the surface. Interfaces involved in transient interactions

are smaller, do not have a significantly different amino acid composition from the rest of the

surface, and may be intrinsically disordered (Perkins et al., 2010).

To fully understand a given protein’s function it must be studied in the context of its interacting

partners. The phenotype of a genomic defect is propagated through the components with which

that gene and its products interact (Barabási et al., 2011). For instance, disruption of protein-

protein interactions are frequently implicated in human diseases. Mutations to the interaction

interface between kinases MKK4 and JNK3, both of which are involved in interleukin signalling

pathways, results in a loss of JNK3 activation and tumor-suppression activity, and is implicated

in adenocarcinoma (Acuner Ozbabacan et al., 2014). Disruption of the formation of multi-protein

complexes has also been observed, as in the case of the RFX (Regulatory Factor X-associated)

complex which controls MHC II expression. Multiple mutations to one member of the complex,

RFXANK, disrupts function, resulting in a loss of MHCII leading to bare lymphocyte syndrome,

a disease characterized by a low levels of immunoglobulin in serum (Wiszniewski et al., 2003).

Disruption of protein-protein interactions with small molecule or peptide inhibitors are also a

valid means of treating human malignancies as, for instance, in the application of cyclic peptides

to disrupt interactions important to the Ras family of oncoproteins (Wu et al., 2013).

Furthermore, the introduction of new, undesired abnormal or erroneous protein-protein

interaction can also induce misfolding in brain proteins or cause aggregation, as seen in

neurodegenerative disorders such as amyotrophic lateral sclerosis, Parkinson’s disease,

Alzheimer’s and various prion diseases (Trojanowski and Lee, 2000).

All interactions within a cell do not occur at the same time and in the same location; spatial and

temporal coordination of the vast majority of cellular processes must occur (Charbonnier et al.,

24

2008). This is achieved through the use of highly-organized, tightly-regulated protein-protein

interaction networks. The cell has a number of mechanisms at its disposal to carry out this

regulation including, but not limited to, regulation of transcription and transcript stability, protein

synthesis and degradation, variation in binding affinity, and post-translational modifications to

affect localization and interactions. Many interactions occur directly between two different

proteins, but some are indirect and require scaffold proteins and other macromolecules to recruit

them and mediate interactions between different members of a given complex.

Interaction networks are intrinsically dynamic, as the majority of protein-protein interactions of

which they are composed are transient and dynamic (Chautard et al., 2009). In order to identify

the interactions of a given protein, a number of techniques may be employed. Yeast Two-hybrid

is one of the more frequently used methods of detecting PPIs, and in theory can detect transient

interactions, but suffers from a high false positive rate (Brückner et al., 2009). Traditional

methods of identifying protein-protein interaction use affinity purification followed by mass

spectrometry (AP-MS). Immunoprecipitation is often ineffective at identifying weak and

transient interactions, often due to wash steps involved in removing non-specific interactions

(Collins and Choudhary, 2008). Given the impact of BCL6 on human disease, the pathways and

protein-protein interactions in which BCL6 and its paralog BCL6B take part are a subject of

intense study, thus the further elucidation of the interaction network of these two homologs may

ultimately provide insight with regards to their role in lymphomagenesis.

In order to address the deficiencies of traditional methods at identifying weak and transient

interactions, complementary methods should be employed to detect a broader range of

interactions for BCL6 and BCL6B in vivo.

1.9 BioID

Proximity-based biotinylation, better known as BioID, is a recently developed method for the

characterization of protein-protein interactions in living cells (Roux et al., 2012). The method

involves the fusion of a modified biotin protein ligase (BirA) from E. coli (Chapman-Smith and

Cronan, 1999a) to a bait protein to biotinylate potential interactors of a protein of interest. Given

that biotinylation is a relatively rare post-translational modification in mammalian cells and is

restricted to a few carboxylases (Chapman-Smith and Cronan, 1999b), specific biotinylation of

potential interactors is useful tool for identification. The modified biotin ligase, henceforth

25

known as BirA*, is fused in-frame to a protein of interest. BirA* contains a mutation, R118G,

that confers a reduced affinity for activated biotin (biotinoyl-5’-AMP). BirA* is able to

efficiently activate biotin, and this highly reactive biotin is then able to diffuse away and react

with proximal amine groups including those present as primary amines in lysine residues of

nearby proteins (Roux et al., 2012). For practical labelling purposes, the radius for biotinylation

by the BioID method in vivo has been identified as ~10nm (Kim et al., 2014). Biotinylated

proteins may then be affinity purified using streptavidin following cell lysis and identified using

mass spectrometry (Figure 1.15).

26

Figure 1.15: Method of BioID protein identification. Fusion protein consisting of BirA* and protein of interest

(Blue and purple protein) is transfected into a cell where it promiscuously biotinylates nearby proteins. The biotin

moiety is represented by red circles, and the radius of effect is shaded red. Cells are lysed and proteins denatured,

and biotinylated proteins are captured streptavidin resin in an affinity purification. These proteins may then be

identified by mass spectrometry. Figure adapted from Roux et al. 2012.

In part because putative interactors are covalently modified, this method is useful as a

complement to traditional Affinity Purification/mass spectrometry interactome methods.

Covalent modification allows the use of harsher lysis techniques, enabling the solubilisation of

27

proteins located in poorly soluble cellular compartments and because protein-protein interactions

do not need to be maintained, during purification weak or transient interactions can be identified

(Coyaud et al., 2015).

1.10 Thesis Objectives

BCL6 deregulation is a potent factor in lymphomagenesis, particularly for diffuse large B-cell

lymphoma. Fully exploring the protein interaction network of BCL6 may give further insight

into the biological roles that BCL6 plays with regards to transcriptional repression. The

interactions between the BCL6 BTB domain and the corepressors NCOR1, SMRT, and BCOR

have already been well-characterized structurally and functionally. These interactions are

important for the role of BCL6 in GC formation and regulation, but do not fully explain all of the

observed phenotypes observed in BCL6 deregulation and BCL6-deficient mouse models. The

use of BioID in particular may identify weak or transient interactions for BCL6 that have not

previously been detected. Additionally, given the high degree of sequence similarity between

BCL6B and BCL6 and the observation that BCL6B interacts with BCL6 to carry out its

repression activity, the possibility exists that BCL6B also plays a role in BCL6 biology and

lymphomagenesis. The protein interaction network of BCL6B is poorly characterized, and it is

one of my objectives to further explore the BCL6B interaction space alongside that of BCL6

using BioID and tandem MS. High-probability interactions will be further validated and

characterized using biochemical methods.

28

2 Materials and Methods

2.1 Cloning and Generation of Constructs

To generate constructs for BioID, full-length cDNA of BCL6 was obtained from Dr. Kevin

Kirouac, full-length cDNA of BCL6B was purchased from Open Biosystems. The pcDNA 5.1

F/R/T Flag-BirA* vector was a gift from the Raut Lab. Quickchange mutagenesis with Pfu

polymerase was used to alter a guanine-339 to an adenine in order to remove an internal NotI site

from the BCL6B cDNA. Mutagenesis was also done on BCL6 cDNA to alter a duplicated PKAC

region at c-terminus also present elsewhere in the BCL6 cDNA. Finally, mutagenesis on BCL6-

PKAC construct was also used to generate the N21K mutant by changing cytosine-63 to guanine.

The modified BCL6 and BCL6B, and N21K cDNAs were amplified by PCR with primers

creating 5’ AscI and 3’ NotI sites. These amplified fragments were subcloned into the pcDNA

5.1 F/R/T plasmid for stable expression in mammalian cell lines. All mutant clones were verified

by sequencing at ACGT corp.

For transient transfection to validate BioID interactions, full-length TLE1, TLE3, and NFIA

cDNAs were purchased from Sparc Biocentre and were provided in the pcDNA 3.1 n-Flag

vector.

The BTB domain (residues 6-136) of BCL6B was cloned into the Pet32a(+) vector (Novagen) by

Neil Pomroy in our lab, the BTB domain (residues 5-129) of BCL6 was cloned into the

pet32a(+) vector by Dr. Farid Ahmad of our lab, the resulting plasmids yield E. coli thioredoxin

protein followed by a linker region containing a 6xHis tag and thrombin cleavage site, with

either BCL6B or BCL6 region following. The BCL6B BTB domain cDNA was further cloned

into the pDuet SUMO FA vector (proper name? who made it?) through amplifying the BTB

domain coding region by PCR with primers that created 5’ AscI and 3’ XhoI sites.

2.2 SDS-PAGE and Western Blots

All gels used were either 8% Tricine gels or NuPAGE Bovex 4-12% Bis-Tris protein gels.

Protein samples were mixed with 2x Laemmli buffer and boiled for five minutes before being

run on gels. For Westerns, proteins were transferred from PAGE gels to Immobilon-P

nitrocellulose membrane (Millipore) using semi-dry method. Membranes were blocked 1 hour in

Odyssey PBS blocking buffer(LI-COR). Primary used antibodies were as follows: Mouse anti-c-

29

Myc (Sigma-Aldrich), Rabbit anti-Flag (Sigma-Aldrich), Mouse D8 anti-BCL6 (Santa Cruz

Biotech), and mouse anti-GAPDH (Life Technologies). Secondary antibodies used were IRDye

680 Goat anti-Mouse IgG (LI-COR), and IRDye 800 Goat anti-Rabbit IgG (LI-COR). Westerns

blots were visualized using Odyssey CLx (LI-COR).

2.3 Mammalian Tissue Culture

HEK293 FlpIn Host cells were cultured in high glucose (4.5g/L) DMEM H21(Wisent)

supplemented with 10% fetal bovine serum and penicillin-streptomycin. Cell were passaged

every 3-4 days under selection with zeocin. HEK293 FlipIn Host cells were cultured to 1.0 x 106

cells/mL in 6-well plates prior and then simultaneously transfected with the following vectors:

1ug of Flag-BirA pcDNA5.1 containing insert of interest (either wtBCL6, BCL6N21K, or

BCL6B) with 3.0ug of pOG44 vector. 24 hours after transfection, cells were trypsinized from 6-

well plates to 10cm plate. 24 hours after trypsinization, selection for cells incorporating the gene

of interest, as well as a hygromycin resistance gene, was begun using 100ug/mL

hygromycin(Wisent). Cell growth and resistance to hygromycin was monitored and media

supplemented with 100ug/mL for several weeks until cell growth became apparent. After cells

had grown to 30-50% confluence, passaging of cell lines was begun and expression of gene-of-

interest was tested by induction with tetracycline (1ug/mL) and Western blot with anti-Flag

antibody.

HEK293 cell lines positive for the gene of interest were expanded to 7x15cm plates. At 2.0x106

cells/mL, expression of BCL6 or BCL6B was induced with 1.0ug/mL tetracycline, and cells

were supplemented with Biotin to a final concentration of 50uM. 24 hours after induction of

expression, cells were harvested in cold PBS with a rubber policeman, washed in cold PBS, then

all media was aspirated and the cells were snap frozen at -80C.

Pellets were lysed in 10mL of modified RIPA lysis buffer (50mM Tris-HCl pH 7.5, 150mM

NaCl, 1mM EDTA, 1mM EGTA, 1% Triton X-100, 0.1% SDS, 1:500 protease inhibitor mixture

(Sigma-Aldrich), 250U Turbonuclease (Accelagen)) at 4˚C for 1h, then sonicated (30s at 35%

power, Sonic Dismembrator 500; Fisher Scientific). Cell lysate was centrifuged at 35000g for

30min. Clarified supernatants were incubated with 30uL packed, pre-equilibrated streptavidin-

Sepharose beads (GE) at 4˚C for 3h. Beads were collected by centrifugation (2000rpm, 2

minutes), washed six times with 50mM ammonium bicarbonate pH 8.3, and treated with TPCK-

30

treated trypsin (Promega). Supernatant containing the tryptic peptides was collected and

lyophilized. Peptides were resuspended in 0.1% formic acid and analyzed by MS.

2.4 Analysis of BioID Results

Mass Spectrometry results were analyzed using the Prohits 4.0.0 suite of software and

Significant Analysis of INTeractome (SAINT) v3.3. Three different experiments were run, each

having a different bait: N-FlagBirA-BCL6, N-FlagBirA-BCL6N21K, or N-FlagBirA-BCL6B, and

samples for each experiment were run in duplicates. For BioID, 18 controls were used for

comparative purposes: twelve runs were conducted on cells expressing the Flag-BirA* tag only,

and six runs conducted on cells expressing Flag-BirA*-GFP. From this analysis, bona fide

interactors were selected as proteins identified with high confidence displaying a SAINT score

≥0.9.

2.5 Transient Transfection and Co-Immunoprecipitation

Flag-tagged “Prey” constructs TLE1, TLE3, NFIA, and SMRT, respectively, were co-transfected

with Myc-tagged “Bait” construct BCL6BTB as follows: 1ug of “bait” construct and 1μg of

“prey” construct were diluted in DMEM H21 and mixed with 3μL of polyjet reagent per μg of

DNA for 15 minutes. The transfection mixture was then added to HEK 293 FlipIn Host cells. 18

hours after transfection, the media was replaced. 24 hours after replacing the media, cells were

harvested for lysis.

During harvesting, cells were washed with cold PBS, then Triton X-100 lysis buffer (20mM Tris

pH8.0, 137mM NaCl, 10% glycerol, 0.1% Triton X-100 detergent, 1mM PMSF, Protease

Inhibitor Cocktail (Sigma) 1:500, and benzonase nuclease (1:1000)) was added to each well,

cells were resuspended in the lysis buffer, then transferred to Eppendorf tubes for 1hour nutation

at 4˚C. Following lysis, cell debris and DNA were spun down at 12000rpm for 20 minutes.

2% of the supernatant volume was set aside in 2X SDS buffer with 1mM BMe to test inputs, and

the rest was added to Pierce anti-c-Myc agarose resin(Thermo Scientific) for binding for 16

hours at 4˚C. Following this, supernatant was removed from the resin, and the sample was

washed three times in cold lysis buffer. Proteins were eluted from the resin by mixing with 2x

SDS buffer (100mM Tris pH 7.0, 4% w/v SDS, 0.2% w/v bromophenol blue, 20% v/v glycerol)

31

and boiled for 5 minutes. Protein-SDS mixture was carefully aspirated to avoid uptaking resin,

and Western blot was used to detect presence of “bait” and “prey” proteins.

2.6 Expression and Purification of BCL6BBTB

E. coli BL21 (DE3) cells were transformed with either the pET32a-BCL6BBTB vector or

pDUET-Sumo FA BCL6BBTB vector via heat shock. Cells were grown at 37˚C in 2L TB media

supplemented with 1% glucose w/v, shaking at 200rpm to and OD600 of 0.6-0.8. Cells were

induced with 0.2mM IPTG and grown 20 hours at 16˚C. Cell cultures were harvested by

centrifugation at 6000rpm and 4˚C. Cell pellets were resuspended in BTB lysis buffer (25mM

tris pH 8.0, 150mM NaCl, 10% glycerol, 10mM BMe, supplemented with EDTA-free protease

inhibitor tablets (1/100mL) (Roche) and benzonase nuclease, and lysed by five passages through

Emulsiflex C5 (Avestin). Cell lysate was centrifuged at 35000rpm for 40 minutes at 4⁰C to

remove cell debris and nucleic acids. Supernatant from centrifugation was passed through 10mL

of NiNTA(Qiagen) that had been equilibrated with BTB buffer (25mM Tris pH8.0, 150mM

NaCl, 10% glycerol) and packed into a column. Flow-through was collected and the column was

washed once with 30mM Imidazole BTB buffer, and then washed again with 70mM Imidazole

BTB buffer to remove non-specific binding of contaminants to the resin. Protein was eluted with

200mM Imidazole BTB elution buffer and collected. Elution fractions were subjected to

overnight dialysis in BTB buffer to remove imidazole.

At this step, Thx-BCL6B fusion protein was set aside for binding assays and a fraction of the

protein was submitted for thrombin cleavage (1U/mg protein) to remove the Thx-6xHis tag.

0.35% Sarcosyl w/v was added to fusion protein alongside thrombin and cleavage occurred

overnight at 4degC. Benzamidine resin (30uL/mg Thrombin) was added to clear thrombin

following cleavage. Samples was incubated with resin for 10min at 4˚C, then centrifuged for

10min at 3800rpm and supernatant was aspirated. Cleaved protein was passed through 10mL of

NiNTA resin and flow-through was collected and concentrated to 3mL. Concentrated protein

sample was run on preparatory size exclusion chromatography Superdex S75 10/300 column

(GE) at 1mL/min flowrate with 2.5mL fractions collected. Fractions identified as containing

BCL6B by SDS-PAGE and Coomassie staining were concentrated to 5mg/mL for use in

crystallization trials.

32

After overnight dialysis, Sumo-BCL6BBTB was subjected to cleavage of the Sumo tag using the

protease ULP1. 6xHis-tagged ULP1 was added to SumoBCL6BBTB at a 1:20 v:v ratio and

samples were left nutating for 16hours at 4˚C. Cleaved Sumo tag and ULP1 protease were

removed from BCL6BBTB by passage through 10mL NiNTA resin, with the flow-through

collected and concentrated to 3mL. Cleaved BCL6BBTB was purified for crystallization as

described above.

2.7 Circular Dichroism of BCL6B BTB in Sarcosyl

CD measurements were obtained using an AVIV 62A-DS CD spectrometer. Ellipticity of the

BTB domain (at 38.8 uM) was measured prior to thermal denaturation at 25˚C from 200nm to

250nM by 1nM steps, in a 1mm cell. Averaging time for each step was 15s, and 17 scans were

performed. All ellipiticity measurements were adjusted for Sarcosyl-BTB buffer (25mM Tris,

50mM NaCl, 10% Glycerol, 10mM BMe, 0.35%w/v Sarcosyl) ellipticity. Thermal denaturation

analyses were carried out using the temperature scan mode and measuring the ellipticity at

216nm. All scans were from 25˚C to 95˚C in one degree steps. Averaging time for each data

point was 20 seconds. Wavelength scans were performed at 95˚C following thermal denaturation

and again at 25˚C following cooling of sample. For thermal denaturation, the fraction of

unfolded protein f was calculated with the equation

f = ([θ216]obs - [θ216]f)/( [θ216]u - [θ216]f), where [θ216]obs is the molar ellipticity at a given

temperature (measured), and [θ216]f and [θ216]u are the molar ellipticity of the fully folded protein

(at 25˚C) and fully unfolded protein (at 95˚C), respectively.

2.8 Microscale Thermophoresis

Measurements for the dissociation constant(KD) of Thx-BCL6BBTB with the SMRT-BBD were

obtained using a Monolith NT.115 (NanoTemper). To obtain determine KD, 10uL of SMRT-

BBD peptide fluorescently labelled with AlexaFluor488 and present at a concentration of

13.75nM was mixed with 10uL of Thx-BCL6BBTB of concentrations ranging from 380μM to

20nM in BTB buffer (25mM Tris pH 8.0, 150mM NaCl, 10% glycerol, 10mM BMe). These

mixtures were incubated for 10 minutes at RT, then aspirated into glass capillaries for

thermophoresis, with each concentration measured at 520nM. Each measurement was performed

at 20˚C for 30s with 40% LED Power and 40% IR power. Scans were repeated with 40% LED

power and 20% IR power for each sample. Experiments were repeated as described above for

33

Thx-BCL6 and Thx-PLZF measurements, except that the concentration of the BCL6 protein

ranged from 188μM to 20nM, and the concentration for PLZF ranged from 258μM to 8nM.

Fluorescence of each measurement was normalized and plotted against the different

concentrations of the titrant protein species. KD fit formula fc = u + (b – u)/2 * F + c + KD –

Sqrt((F + c + KD)^2 – 4*F*c) was used to calculate the KD value with the GraphPad Prism

software, where F is the concentration of the labelled SMRT-488 peptide and c is the

concentration of the titrant protein.

2.9 Crystallization of BCL6BBTB

Crystal trials were set up using the NT8 robotics platform (formulatrix). For screening, JCSG

Core Suite I-IV (Qiagen) was used with 96-well intelliplates (ArtRobbins Instruments). Drops

were set up with 200nL of protein solution mixed with 100nL of precipitant solution. Trays were

covered and placed in RockMaker Imager (Formulatrix) for imaging.

2.10 Phylogenetic Analysis and Sequence Conservation

Protein sequences of BCL6 and BCL6B respectively, as identified in Homo sapiens, were

collected from Uniprot database and used as the basis for identifying orthologs in the following

species: Mus musculus, Bos taurus, Ornithorhynchus anatinus, Monodelphis domestica, Gallus

gallus, Taeniopygia guttata, Chrysemys picta belii, Anolis carolinensis, Xenopus tropicalis,

Danio rerio, Takifugu rubripes, Latimeria chalumnae, Callorhincus milii, Lethenteron

japonicum, and Ciona intestinalis. The genomes of these species were examined using BLASTP

and querying the RefSeq and SwissProt databases. In order to identify unannotated Lamprey

orthologs, the Japanese Lamprey database was queried using BLASTN and TBLASTN searches

with L. chalumane BCL6/B orthologs as the query sequences. Identified exons were collated to

form the BCL6B sequence. All amino acid sequences were aligned using MUSCLE.

Phylogenetic trees were generated with PhyML using the maximum likelihood method with 100-

bootstrap runs.

34

The Protein Variability Server (PVS—Garcia-Boronat et al. 2008) was used for sequence

variability analysis by Shannon Diversity of the given BCL6 and BCL6B multiple sequence

alignments. A 3D structure for the BCL6B BTB domain was generated by homology modelling

using the iTasser web server (Zhang 2008). Sequence entropy values were mapped onto the PDB

structure entry 1R28 for BCL6 or the generated BCL6B BTB domain model. Co-evolution of

dimerization-mediating residues for the BCL6 and BCL6B BTB domains were predicted using

the GREMLIN contact prediction method webserver, highlighting pairs of residues likely to be

in contact with each other in the 3-dimensional BTB structure (Kamisetty et al. 2013).

35

3 Results

3.1 BioID identifies known and novel interactors of BCL6 and BCL6B

Our first goal was to identify interactors of BCL6 and BCL6B in vivo, and so it was reasoned

that using the BioID method would result in the labelling of proximal interacting partners with

biotin. These interactions could then be identified by streptavidin pull-down and mass

spectrometry. Towards this end, a FlagBirA* tag was fused to the N-terminus of BCL6,

BCL6N21K, and BCL6B to generate FlagBirA*-BCL6, FlagBirA*-BCL6 N21K, and FlagBirA*-

BCL6B, respectively, and these proteins were stably expressed in 293 T-Rex Flp-In cells. The

different FlagBirA*-fusion proteins were expressed in a tetracycline-dependent manner and

biotin was added to the culture media upon induction of expression. Cells were lysed using a

modified RIPA buffer followed by sonication and nuclease treatment to maximize the

solubilisation of proteins. An aliquot of the cellular lysate was used to detect the presence of the

Flag-BirA* fusion proteins by western blot using an anti-flag antibody.

Biotin-labelled proteins were captured using a streptavidin-sepharose matrix. After washing,

streptavidin-bound proteins were treated with trypsin, and the eluted peptides were identified

using nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-

ESI-MS/MS). Cells expressing FlagBirA* tag only (12 samples) or FlagBirA*-tagged GFP (6

samples) were subjected to the same analysis as controls for endogenously biotinylated proteins

and polypeptides that interact nonspecifically with the streptavidin or sepharose solid phase

support.

Using the ProHits system (Liu et al., 2010), MS data were analyzed with the X!Tandem database

search algorithm (Craig and Beavis, 2004) and the resulting peptides identified were subjected to

Significance Analysis of INTeractome (SAINT) statistical analysis (Nesvizhskii et al., 2003)

compared against control sets subjected to the same protocol for expression, lysis, and analysis.

Polypeptides assigned a SAINT score ≥0.9 were identified as bona fide interactors with BCL6

(Table 1) and BCL6B (Table 2).

36

Table 3-1: Significant interactors for BCL6 identified by BioID. This table contains the

SAINT scores and peptide counts for each protein hit identified by BioID. Columns 3 and 4

show the peptide counts for each of the two replicates. Column 5 lists the sum of spectral counts

from the 18 control experiments.

Hit SAINT Score V1 V2 Control

NCOR1 1 375 386 0

BCOR 1 253 232 3

NCOR2 1 169 173 0

TBL1XR1 1 110 109 0

HIST1H4E 1 94 103 0

HIST1H2AJ 1 71 65 0

TLE3 1 49 50 3

TBL1X 1 43 37 0

NFIA 1 40 36 5

KDM2B 1 29 34 0

TLE1 1 34 27 0

GPS2 1 28 27 0

HIST1H2AA 0.92 25 27 14

HIST1H3D 1 25 24 0

H2AFZ 0.9 23 24 0

ZNF318 1 23 21 4

37

TCF20 1 16 26 0

HDAC3 1 19 18 3

NFIB 1 18 19 0

VDAC1 1 15 13 0

NFIC 0.97 13 14 0

C11orf49 1 9 9 0

NFIX 1 9 9 0

PCGF1 1 7 11 0

BCL6B 1 8 9 0

KIAA0182 1 7 8 0

MMP24 1 5 8 0

CPVL 1 5 6 0

FOXK1 1 4 5 0

DLD 0.95 3 5 0

KDM1A 0.94 4 3 0

ANP32A 0.96 2 4 0

POLR2H 0.99 3 3 0

AES 0.91 2 2 0

DPEP1 0.91 2 2 0

38

NKX2-1 0.91 2 2 0

PPIH 0.91 2 2 0

TM2D3 0.91 2 2 0

Table 3-2: Significant interactors for BCL6B identified by BioID. This table contains the

SAINT scores and peptide counts for each protein hit identified by BioID. Columns 3 and 4

show the peptide counts for each of the two replicates. Column 5 lists the sum of spectral counts

from the 18 control experiments.

Hit SAINT Score V1 V2 Controls

NCOR1 1 239 234 0

HIST1H4E 1 124 204 0

HIST1H2BB 1 128 165 43

NCOR2 1 147 141 0

BCOR 1 79 87 3

TBL1XR1 1 78 75 0

SIN3B 1 61 68 25

ARID3B 1 56 63 8

CENPC1 1 62 54 0

C1QBP 1 52 57 16

DACH1 1 45 47 2

ZNF281 1 42 49 5

39

NFIA 1 44 41 5

ZBTB9 1 44 40 0

TFAP2A 1 42 34 0

RPS24 1 32 35 9

TLE3 1 33 33 3

HIST1H3D 1 28 30 0

ZNF318 1 27 29 4

TBL1X 1 24 25 0

WIZ 1 21 22 0

GPS2 1 18 24 0

RPL27 1 21 21 2

NACC1 1 21 20 7

NFIB 1 17 23 0

AP2M1 1 20 19 4

SLU7 1 18 20 6

VDAC1 1 18 19 0

NFIC 1 19 16 0

KDM1A 1 16 16 0

PPIL3 1 17 13 0

40

TLE1 1 13 15 0

TFAP2C 1 11 12 0

ZNF536 1 11 11 0

SULT1A1 1 10 9 0

NFIX 1 9 9 0

COIL 1 6 11 0

CPVL 1 8 9 0

KDM2B 1 8 9 0

RPS10-

NUDT3

1 8 9 0

OAT 1 6 9 0

TCF20 1 7 8 0

ZEB2 1 9 6 0

BCL11A 1 5 5 0

KIAA0182 1 6 3 0

C16orf88 1 4 4 0

FLI1 1 4 4 0

FOXP1 1 5 3 0

TSHZ3 1 5 3 0

HIST2H2AB 0.99 52 38 0

41

ZMYM2 0.99 6 4 0

FOXJ3 0.99 3 4 0

FOXK1 0.99 4 3 0

HOXC13 0.99 4 3 0

PCGF1 0.99 3 3 0

POLR2H 0.99 3 3 0

ZKSCAN4 0.99 3 3 0

H2AFZ 0.98 39 37 0

HDAC3 0.98 11 12 3

SSR4 0.97 10 8 2

ZNF131 0.97 6 8 0

TCF7L1 0.97 2 5 0

MGA 0.97 2 3 0

NKX2-1 0.97 2 3 0

PHF8 0.97 3 2 0

PRR12 0.97 3 2 0

ZNF174 0.97 3 2 0

C11orf49 0.96 3 5 0

TEX10 0.96 4 3 0

42

TULP3 0.94 2 2 0

LIN54 0.93 6 6 0

PCNA 0.92 17 20 6

DHX40 0.92 5 5 0

Of the 641 unique polypeptide interactors identified in the unfiltered BCL6 dataset 43 had a

SAINT score greater than 0.9. For the unfiltered BCL6B dataset, 735 interactors were identified

and from these 78 had a SAINT score of 0.9 or greater. A further six interactors were removed

from BCL6, and three from BCL6B by filtering using the Contaminant Repository for Affinity

Purification (CRAPome) database (Mellacheruvu et al., 2013).

The list of BCL6 specific interactors included many previously identified binding partners:

notably SMRT and NCOR1, and associated members of the SMRT/NCOR1 complex TBL1X,

TBL1XR1, GPS2, and HDAC3; and BCOR and associated PRC1-like complex members PCGF1

and KDM2B. In addition, thirteen previously known interactors or members of interacting

complexes were identified, while twenty-four were novel interactions. All seventy-five of the

interactors identified for BCL6B were novel interactors not previously identified in the literature.

Between the two datasets, 27 putative interactors were identified with both the BCL6 and

BCL6B baits. SMRT, NCOR1, BCOR, and members of the SMRT/NCOR1 repression complex

and PRC1-like complex, respectively, were among these shared interactors. The majority of

identified interactors, 32 for BCL6 and 63 for BCL6B, are annotated as nuclear proteins, which

is consistent with the localization of BCL6 and BCL6B. Additionally, 26 and 37 proteins for

BCL6 and BCL6B, respectively, were annotated as having DNA-binding activity, and of these

proteins, ten from the BCL6 dataset and 28 from the BCL6B dataset were noted as transcription

factors.

The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING—von Mering et al.,

2003) was used to analyse interactions within the protein sets (Figures 3.1A and B). Individual

43

proteins are represented as nodes, and edges represent interactions. Nodes are coloured according

to the functional module to which they belong, generated using the MCL clustering feature.

Several clusters are apparent in each interaction map. For both BCL6 and BCL6B datasets, a

significant number of the interactions are centered on HDAC3. However, nearly half the nodes

are not connected to other proteins in the lists, and most of these are transcription factors.

A)

A

)

44

B)

Figure 3.1: STRING Network of Interactors identified in BioID experiments. A) Identified BCL6 interactors.

The list of putative interactors identified as statistically significant in BCL6 BioID experiments was given to

STRING as input. Each protein is represented as a circular node, with larger nodes representing protein for which a

structure has been solved or generated by homology. Previous interactions identified in the literature are represented

as edges connecting nodes, with different-coloured edges representing different evidence of interaction. Solid edges

represent interactions within a cluster as determined by MCL, dotted edges represent interactions between nodes in

different clusters. Nodes within the same complex cluster share colours, nodes with no shared colour were not found

to cluster with other nodes in this dataset. The yellow cluster represents histone-associated proteins including

members of the NCOR1/SMRT repression complex and nucleosome; brown nodes are part of the PRC1-like cluster;

purple nodes are associated with the CTF-NFI transcription factor family; orange nodes are ribosomal subunits;

connected red nodes are BCL6 and BCL6B. B): STRING Network of Interactors identified in BCL6B BioID

experiments. The input given to STRING is the list of putative interactors identified as statistically significant in

BCL6B BioID experiments. The orange cluster represents histone-associated proteins including members of the

NCOR1/SMRT repression complex; brown nodes represent nucleosomal proteins; yellow nodes are part of the

PRC1-like cluster; pink nodes are associated with the CTF-NFI transcription factor family; purple nodes are

45

ribosomal subunits; light blue nodes are members of the groucho/transducin-like enhancer of split complex; red

nodes are associated with the Lysine Demethylase 1 (LSD1) complex; light green nodes play a role in protein

folding.

Amongst the clusters identified in the interaction network, the NCOR1/SMRT repression

complex and PRC1-like complex are prominent. Additional clusters consist of core histone

proteins, ribosomal protein subunits, the CTF/NFI family of transcription factors, and members

of the groucho/transducin-like enhancer family of corepressors. Because interactions from these

latter two groups of proteins are not described in the literature, these proteins were chosen as

candidates for validation and further characterization.

3.2 Validation of Identified Interactions

Based on their annotated functions as corepressors, peptide count and coverage, and co-

expression with BCL6, Transducin-like Enhancer of Split 1 and 3 (TLE1 and TLE3) were

selected for further characterization. Nuclear Factor I A (NFIA) was also selected as a candidate

for validation as a representative of the CTF/NFI family based on peptide count and coverage,

and its role as a transcription factor mediating repression of gene expression.

Constructs were generated for mammalian expression of TLE1, TLE3, and NFIA using the

pcDNA 3.1 N-FLAG vector. These constructs were transfected into 293 T-Rex FlpIn cells stably

expressing FB-BirA* BCL6, the expression of which was induced simultaneously with the

transfection. These experiments failed to produce an interaction with a SMRT-BBD positive

control (SMRT1) construct detected by CoIP. To overcome this, a pcDNA3.1 N-myc construct

containing the BTB domain (residues 5-129) of BCL6 was created for use as a bait, and co-

transfecte with each separate FLAG-tagged candidate prey construct in HEK293 cells.

These cells were lysed using Triton X-100 lysis buffer and extracts were immunoprecipitated

with Myc-agarose resin and analyzed by anti-FLAG and anti-myc Western blots. Two fragments

of SMRT, FLAGSMRT1 (which contains the BBD) and FLAGSMRT4 (which does not contain the

BBD), were included as positive and negative controls, respectively. No interaction was detected

between the BTB domain of BCL6 and NFIA by Western blot, while both TLE1 and TLE3 were

detected (Figure 3.2)

46

Figure 3.2: Western Blot of Inputs and BCL6 interaction validation by CoIP. FLAG-tagged SMRT1 (positive

control), TLE1, TLE3, and SMRT4 (negative control), respectively, were co-transfected with myc-tagged BCL6BTB.

Proteins were visualized by Western blot using anti-myc and anti-FLAG antibodies respectively. The top panel

contains flag-tagged Prey bands, the bottom panel contains myc-tagged BCL6 bait bands.

3.3 Purification of the BCL6B BTB domain

3.3.1 Constructs and Purification Strategy

The BCL6B BTB domain, comprising residues 6-136, was fused to an N-terminal 6xhis-

thioredoxin tag in pET32a(+) vector for E. coli expression. This expression yielded 12-

32mg/litre of E. coli culture. Purification of the BCL6B BTB domain consisted of four

chromatographic steps, beginning with Ni-NTA chromatography, followed by either gel

filtration or dialysis, then thrombin protease cleavage, an additional Ni-NTA column to remove

the cleaved tag, an then a final gel filtration step, yielding 0.066mg/litre of pure protein (Figures

3.3 and 3.4). Thioredoxin-BCL6BBTB eluted at twice the expected size by gel filtration,

supporting a dimeric structure for the fusion protein. Following the thrombin cleavage step and

removal of the thioredoxin tag, BCL6B is poorly soluble and forms a visible precipitate.

47

Inclusion of residues flanking the BTB domain at its C-terminus was attempted, but these

constructs behaved similarly during purification. Likewise, attempts to alter the buffer

composition (e.g. changing salt concentration, buffer pH, or buffer system) gave negligible

improvements to the solubility. To overcome this, the anionic detergent sarcosyl was used as an

additive to improve solubility, using concentrations between 0.1% and 0.35% weight by volume

(Figure 3.5). At 0.1% w/v, sarcosyl improved the yield of pure BCL6BBTB to 2-4mg/litre.

Following the addition of sarcosyl, it was observed that off-target cleavage was occurring during

the thrombin protease cleavage step. To overcome this, BCL6BBTB was cloned into a pDUET-

SUMO FA vector, replacing the N-terminal 6xHis-Thioredoxin tag with a 6xHis-SUMO tag.

This allowed for the use of a structure dependent protease, ULP-1, which recognizes the tertiary

structure of the SUMO tag, as opposed to the sequence-dependent recognition and cleavage by

thrombin, while maintaining the same purification strategy (Figure 3.6).

Figure 3.3: SDS-PAGE gel showing the purification of Thx-BCL6BBTB by Nickel Affinity Chromatography.

Lane 1 = molecular weight markers, Lys = cell lysate supernatant following high-speed spin, FT = flow-through

from nickel resin, W10 and W30 = fraction of wash with 10mM Imidazole buffer and 30mM imidazole buffer,

respectively, 1-5 = fractions containing Thx-BCL6BBTB eluted with 200mM imidazole BTB buffer.

48

Figure 3.4: Gel filtration profile of Thioredoxin-BCL6BBTB. Chromatography was performed in a buffer

composed 20mM Tris, 150mM NaCl, 10% glycerol v/v and 10mM βme, run on a Superdex XK16 S-75 column (GE

Healthcare).

Figure 3.5: Structure of the sarcosyl anionic detergent molecule. The critical micelle concentration of sarcosyl

is 0.4% w/v.

49

Figure 3.6: SDS-PAGE gel showing the purification of BCL6BBTB following cleavage of 6xHis-SUMO tag.

Lane 1 = BCL6BBTB sample immediately after cleavage with ULP1, 2 = input onto nickel resin column, 3 = flow-

through from nickel resin column, 4 = pooled fractions eluted with 10mM imidazole, 5 = concentration of pooled

flow-through and elution fractions containing BCL6BBTB.

3.3.2 Evaluation of stability, solubility, and folding of BCL6BBTB in the presence of Sarcosyl

The addition of sarcosyl may serve to solubilize BCL6B by denaturing it. To further determine if

BCL6B is soluble and well-folded in the presence of sarcosyl, circular dichroism spectroscopy

was used to measure secondary structure and thermal stability. CD spectra were measured for a

sample of purified BCL6BBTB at 25⁰C and normalized against a background of sarcosyl-

containing buffer. Cleaved BCL6B showed α-helical and β-sheet characteristics (Figure 3.7)

with strong ellipticity at 216nm. Ellipticity was recorded at 216nm as a function of temperature

over the range of 25-95⁰C (Figure 3.8). A spectrum was measured at 95⁰C and again at 25⁰C

following cooling to assess whether the structural changes to BCL6BBTB were reversible. In the

presence of sarcosyl, BCL6BBTB had a midpoint transition temperature (Tm) of 78⁰C. These

spectra show the presence of α-helical and β-sheet secondary structure elements prior to

denaturation, and loss of these elements following denaturation. This indicates that BCL6B

adopts a folded structure with distinct secondary structure elements with the addition of sarcosyl.

50

Combined with size exclusion data indicating BCLCB forms a dimer in the presence of sarcosyl,

these results suggest that the BTB domain is both stable and well-folded when 0.35% w/v

sarcosyl detergent is used as a solubilizing additive.

Figure 3.7: Circular Dichroism spectra of BCL6B protein. CD was performed in Tris buffer pH 8.0 with

0.35% sarcosyl additive. Overlap of BCL6B spectrum at 25⁰C (blue), 95⁰C during temperature melt (orange), and

25⁰C following temperature melt (grey).

51

Figure 3.8: Temperature profile of BCL6B. Temperature melt was performed in Tris buffer pH 8.0 with 0.35%

sarcosyl as measured by circular dichroism at 216nm. Temperature was increased by 1⁰C every 20 seconds to

generate the temperature profile. The sigmoidal curve indicates cooperative unfolding with a transition point at

78⁰C.

3.3.3 Crystallization Trials

The use of sarcosyl led to yields of pure BCL6B BTB domain of approximately 2-4 mg/L

following size exclusion chromatography, where the protein elutes as a dimer (Figure 3.9). Pure

protein samples were concentrated to 2mg/mL and 3mg/mL, and put into crystal trials with

JCSG commercial screens (Qiagen). A condition of 0.5% w/v of sarcosyl in BTB purification

buffer was used as a blank control for crystal formation. Crystals were observed in a number of

conditions, appearing as rhomboid plates that extinguish polarized light, however, these crystals

were identified as salt based on their diffraction patterns. Identification of crystallization

conditions from which to continue optimization is ongoing.

Tm=78˚C

52

Figure 3.9: Gel filtration profile of BCL6BBTB run on an se70 analytical column (BioRad). Void volume and

molecular weight of the peak (in kDa) are indicated.

3.4 Biophysical Characterization of the interaction between BCL6B and SMRT

Microscale thermophoresis (MST) was chosen as a quantitative technique to further examine the

interaction between BCL6B and SMRT due to its label-free nature and low sample consumption.

MST works on the principle of thermophoresis: the directed movement of particles in a

temperature gradient. This directed movement is dependent on the interface between molecules

in solution and the solvent, and can be affected by size, charge, and solvation entropy of the

53

molecules. The thermophoresis of an individual protein typically differs significantly from the

thermophoresis of a protein-ligand complex (Seidel et al., 2012).

To evaluate complex formation, a SMRT peptide (residues 1414-1430) representing the minimal

binding region of SMRT to the BCL6 BTB domain was obtained. This peptide was fluorescently

labelled with AlexFluor488 dye with an excitation maximum at 490 nm and an emission

maximum at 525 nm. The Thx-BCL6 BTB domain was used as a positive control and Thx-

PLZFBTB, another BTB-ZF protein with no SMRT BBD-binding affinity was used as a negative

control. The results of MST experiments shows binding between Thx-BCL6B and SMRT1414-

1430 (Figure 3.10A) with a fitted KD of 10.9μM. This is approximately tenfold greater than the

KD of 0.984μM measured for BCL6 (Figure 3.10B). The KD measured for BCL6 is comparable

with values in the literature of 1.11μM obtained by surface plasmon resonance, and 11.4μM

obtained by isothermal titration calorimetry. No interaction was measured between Thx-PLZFBTB

and the SMRT peptide.

54

Figure 3.10: SMRT-BBD-BTB domain interaction binding curves A) Binding curve of Thx-BCL6B and

SMRT-488 peptide determined by microscale thermophoresis. SMRT-488 concentration was kept constant at

13.75nM while BCL6B concentration increased from 10nM to 380μM. SMRT peptide showed an increase in

fluorescence from the bound to the unbound state. The fitted KD is 10.9μM as determined by Monolith NT.115

analysis software. B) Binding curve of Thx-BCL6 and SMRT-488 peptide determined by microscale

thermophoresis. SMRT-488 concentration was kept constant at 13.75nM while BCL6B concentration increased

from 20nM to 23.5μM. SMRT peptide showed an increase in fluorescence from the bound to the unbound state. The

fitted KD is 0.984μM as determined by Monolith NT.115 analysis software.

55

3.5 Bioinformatic Analyses

3.5.1 Phylogenetic analysis of BCL6/B

Due to the presence of NCOR1/SMRT and BCOR as potential interactors for BCL6B as well as

BCL6, phylogenetic analysis was done to probe the evolutionary conservation of BCL6 and

BCL6B and potentially identify where these two proteins diverged, as well as to probe the rates

of evolution and sequence conservation within these proteins. Furthermore, the interactions

between BCL6 and NCOR1, SMRT, and BCOR are important in adaptive immunity, given their

role in the germinal centre reaction. Adaptive immunity has only been observed in vertebrates,

and only jawed vertebrates possess immunoglobulin-based immunity. Jawless vertebrates, which

are the most phylogenetically distinct vertebrates from mammals, possess an alternative form of

adaptive immune system that uses leucine rich repeat motifs for antigen recognition. Thus,

identifying the conserved evolution of the BCL6 complex may elaborate on the functions of

BCL6 and BCL6B.

The organisms chosen for this analysis represent a selection of jawed vertebrates (gnathostomes)

from different orders whose genomes have been sequenced, as well as the Japanese lamprey, a

jawless vertebrate (agnathan), and the tunicate, which lacks an adaptive immune response

(Cooper and Alder, 2006).

Sequence database queries from RefSeq, SwissProt, and the Japanese Lamprey database

identified a number of BCL6/B homologs in each of the organisms chosen. These sequences

were annotated according to species and query protein used to identify them from the database

(BCL6 or BCL6B) and aligned using the multiple sequence alignment tool MUSCLE (Edgar,

2004). From these alignments, the sections of each sequence representing the BTB domain,

middle linker region, and zinc finger domains were also identified, and phylogenetic analysis

was performed with inputs of full-length protein sequences, BTB domains, and zinc fingers

(Supplementary figures). Phylogenetic trees were generated from phyML (Guindon et al., 2010)

data using the FigTree graphical viewer for phylogenetic trees (http://www.atgc-

montpellier.fr/phyml/). Analysis of these phylogenies revealed two distinct groups, one

corresponding to BCL6 and one corresponding to BCL6B. The BCL6AB proteins identified in

ray-finned fish were located within the BCL6 group. Comparison of the two groups showed a

http://www.atgc-montpellier.fr/phyml/

http://www.atgc-montpellier.fr/phyml/

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more rapid rate of change in the BCL6B group relative to the BCL6 group. Loss of BCL6B was

observed in several lineages, whereas BCL6 was found in all of the selected jawed vertebrates.

BCL6B was notably absent from birds, lizards, and frogs, and the ghost shark, an early jawed

vertebrate. The homologs in the Japanese lamprey and tunicate were more closely related to

BCL6B than BCL6. Homologs identified for phylogenetic study and their classification are

summarized in Table 3 and displaying these species with their positions in the tree of life is

displayed in Figure 3.11. A representative tree showing the two separate groups was generated

using sequences for the BTB domain identified for each protein, in Figure 3.12; additional trees

comparing the zinc finger regions and full-length BCL6 and BCL6B proteins can be found in the

appendices.

Table 3-3: Summary of BCL6/B homologs identified in representative organisms. Species

identifier is given in the first column and colloquial name and two-letter identifier in the second

column. Columns 3-5 show whether a given BCL6, BCL6B, or BCL6AB homolog is present in a

given organism.

Species

BCL6 BCL6B BCL6AB

Homo sapiens Hs Human yes yes no

Mus musculus Mm Mouse yes yes no

Gallus gallus Gg Chicken yes no no

Taeniopygia guttata Tg Zebra finch yes no no

Chryemys picta belii Cp Turtle yes yes no

Anolis carolinensis Ac Anole yes no no

Xenopus tropicalis Xt Frog yes no no

Danio rerio Dr Zebrafish yes yes yes

Takifugu rubripes Tr Pufferfish yes yes yes

Latimeria chalumnae Lc Coelecanth yes yes no

Calorhincus milii Cm Ghost shark No yes no

Lethenteron japonicum Lj Japanese lamprey No yes no

Ciona intestinalis Ci Tunicate Yes no no

57

Figure 3.11: Phylogenetic representation of query species and presence of BCL6/B homologs. A

phylogenetic tree, based on the tree of life, displaying the query organisms used in phylogenetic analysis, as well as

which BCL6/B homologs are present in that species. BCL6 is shown as a dark blue dot, BCL6B as a light blue dot,

and BCL6AB as a yellow dot near the name of the species. Proposed gene duplication (green triangle) and gene

deletion (red square) events are also shown.

59

Figure 3.12: Phylogenetic Tree of BTB domains: A) Rooted phylogenetic tree generated using BTB domains of

human BTB-ZF proteins, with drosophila BTB domains as an outgroup. Sequences were aligned using MUSCLE

and the tree was generated by FigTree (v 1.4). BCL6/B BTB domains of different vertebrates were included in the

alignment and are highlighted in red (BCL6) and blue (BCL6B). The number at each of the nodes represent

bootstrap values (as a % of trees generated). B) BCL6 and BCL6B BTB domains expanded from 3.12A

60

3.5.2 Conservation of Corepressor BBDs

The selected model organisms were also used to evaluate the conservation of the BBD motif in

SMRT, NCOR1, and BCOR. These sequences were aligned by MUSCLE and visualized using

JalViewer (Figure 3.13). The NCOR1 BBD was found in all species of vertebrate for which it

was queried, including both Japanese lamprey and sea lamprey. Neither SMRT nor BCOR were

present in ray-finned fish species (zebrafish and pufferfish) and agnathans (Japanese and sea

lamprey) (Table 4). All three BBDs are extremely well-conserved within the organisms in which

they are present. Exploration for different members of the NCOR1/SMRT repression complex

and PRC1-like complex was also performed and showed similar conservation within vertebrates

to the corepressor proteins that mediate complex formation (Supplementary Figures).

3.5.3 Conservation of the BCL6 and BCL6B lateral grooves

As the BCL6-BBD interaction is mediated through the lateral groove of the BTB domain, the

level of conservation of residues within the lateral groove might provide a point of comparison

between BCL6 and BCL6B. To this end, Shannon entropy, a statistical measure of conservation,

was applied to multiple sequence alignments of the BTB domains of BCL6 and BCL6B

respectively. Shannon entropy values were then mapped onto the structure of BCL6, obtained

from the PDB entry 1R29, and a model of the BCL6B generated through homology by the

iTasser web server (Figure 3.14).

This analysis revealed that the lateral groove and dimerization interface of BCL6 are extremely

well-conserved among jawed vertebrates. Entropy values for nearly all of the residues

comprising these regions were zero, indicating complete conservation. In BCL6B, these regions

were slightly less well-conserved. The area comprising the dimerization interface of BCL6B is

nearly as well-conserved as that of BCL6, with the majority of residues having entropy values

near zero. Residues comprising the lateral groove have much higher variance, in particular

Leucine-28 and Glutamate-30, which appear to differ in nearly every ortholog of BCL6B.

Residues Asparagine-27 and Histidine-122, analogous to N21 and H116 in BCL6, show full

conservation amongst all BCL6B sequences.

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Figure 3.13: Graphical representations of the sequence alignments of BBDs from NCOR1, SMRT, and

BCOR. Residues are coloured by type, with the consensus sequence presented below the alignment. The BBD for

each protein sequence is bracketed in black lines and shown in pink. Hs = human, Mm= mouse, Gg = chicken, Tg =

zebra finch, Ac = anole, Cp = turtle, Xt = frog, Dr = zebrafish, Tr = pufferfish, Lc = coelacanth, Cm = ghost shark,

Lj = Japanese lamprey, Pm = sea lamprey.

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Table 3-4: Summary of NCOR1, SMRT, and BCOR homologs identified in representative

organisms. Species identifier is given in the first column and colloquial name and two-letter

identifier in the second column. Coloured columns show whether a homolog is present in a given

organism and also whether the BBD is found in that organism. SMRT and NCOR are coloured in

orange, BCOR is coloured in green.

NCOR1 SMRT BCOR

Species

Full length BBD

Full length BBD

Full length BBD

Homo sapiens Hs Human Yes yes yes yes yes yes

Mus musculus Mm Mouse Yes yes yes yes yes yes

Gallus gallus Gg Chicken Yes yes yes yes yes yes

Taeniopygia guttata Tg Zebra finch Yes yes yes yes yes yes

Chryemys picta belii Cp Turtle Yes yes yes yes yes yes

Anolis carolinensis Ac Anole Yes yes yes yes yes yes

Xenopus tropicalis Xt Frog Yes yes yes yes yes yes

Danio rerio Dr Zebrafish Yes yes yes yes yes yes

Takifugu rubripes Tr Pufferfish Yes yes yes yes yes yes

Latimeria chalumnae Lc Coelecanth Yes yes yes yes yes yes

Calorhincus milii Cm Ghost shark Yes yes yes yes yes yes

Lethenteron japonicum Lj

Japanese lamprey Yes yes no no no no

Ciona intestinalis Ci Tunicate No no yes no yes no

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Figure 3.14: Shannon sequence entropy mapped to the surface of the BCL6 and BCL6B BTB domain

monomers. Sequence entropy was calculated from MUSCLE multiple sequence alignment. BTB domains are

presented showing two views: a front view showing the lateral groove and dimerization interface, and a top view.

64

3.5.4 Co-evolving Residues in the BTB Domain

BioID experiments showed that both BCL6 and BCL6B interact with other transcription factors,

including other members of the BTB-ZF family. Some members of the BTB domain are able to

form heterodimers with each other to further expand their activity; among these it has been

posited that BCL6 and BCL6B are able to heterodimerize and that it might be necessary for this

to occur in order for BCL6B to exhibit repression activity (Takenaga et al., 2003). The BCL6

BTB domain can form heterodimers with the Miz1 BTB domain (Stead and Wright, 2014). As

both BCL6 and BCL6B are obligate homodimers, and based on results from entropy analysis of

the BTB domain of both proteins, we propose that the contacts between residues necessary for

the formation of BTB dimers will be well-conserved, and thus that these residues will co-evolve.

I therefore made use of GREMLIN, a method that uses large numbers of multiple sequence

alignments to predict strongly co-evolving residues by way of a global statistical model

(Kamisetty et al., 2013).

The BTB sequences of BCL6 (residues 5-129) and BCL6B (6-136) were input to GREMLIN,

which used these as seeds to generate large multiple sequence alignments from sequence

databases (8894 sequences for BCL6 and 7113 for BCL6B. Covarying residues were overlaid on

a structure based contact map from the top ten closest homologous sequences found in the PDB

(Figure 3.15).

From these predicted co-evolving residues, three pairs were predicted as inter-oligomer contacts

in BCL6 and four in BCL6B. Within BCL6, I30 and Y58, L25 and T48, and N21 and C53,

respectively, are predicted to strongly co-evolve. Within BCL6B, I36 and F62, L31 and A54,

N27 and C59, and I36 and R68, respectively, are predicted to strongly co-evolve. An alignment

of the BCL6 and BCL6B BTB domains shows that these residue positions are analogous

between the two proteins and most of the BTB domains. (Figure 3.16).

The dimer structures of these two BTB domains, 1R28 for BCL6 and the homology model

generated for BCL6B, show that these pairs of residues are in close proximity with each other

(Figures 3.17). In both BCL6 and BCL6B, one member of each pair is found as part of the α1

secondary structure element and the other member as part of either α2 or α3 located at the

dimerization interface. Furthermore, the side chains of these residues are oriented towards their

65

predicted co-evolving residue. Some of these co-evolving residues are found as part of the lateral

groove forming contacts with the BBD, notably BCL6 N21 and C53 (BCL6B N27 and C59).

A) BCL6

Figure 3.15: Contact map of co-varying BTB residues. Shown as left to right and top to bottom show the

residues from N to C terminus. The top 60 co-evolving residues within the BTB domain of BCL6 (A) and BCL6B

(B), overlaid on residue contacts from the top 10 most similar structures to the query sequence, by homology, from

the pdb database. The darker and larger the blue dots, the greater the covariance. The shade of the underlying circles

is an average of those ten structures, red shaded circles represent inter-oligomer contacts.

Figure 3.16: Sequence alignment of the BCL6 and BCL6B BTB domains with co-evolving inter-dimer

contact residues highlighted and connected to their “co-evolving” partner residue by arcing lines. Secondary

66

structure elements of the BTB domain are shown as green bars (α-helices) and blue arrows (β-sheets). Conserved

residues are denoted by a star, similar residues by dots.

Figure 3.17: Location of co-evolving residues in the BCL6 BTB homodimer. A) Pairs of interacting residues

are coloured the same (yellow, orange, or red) and the exact residue type and number, along with the probability of

their co-evolving as determined by GREMLIN, is provided. B) The zoomed in view shows the C-terminus of α1

from one BTB domain and α2 and α3 from the other. Co-evolving residues are labelled and coloured as in part A).

67

4 Discussion

4.1 Experimental Rationale

Previous exploration of the interactome of BCL6 has been undertaken using affinity purification

to pull down interactors in different cellular environments (Hu, 2010; Miles et al., 2005). The

nature of these interactome studies is biased towards the identification of stronger interactions

and may have missed biologically relevant interactions that are more easily disrupted. In an

effort to capture these interactions, we made use of BioID, a proximity dependent-labelling

method capable of detecting proximal interactors by biotinylation. We applied this technique to

the interactions of BCL6 and its close ortholog BCL6B, which shares a high degree of sequence

similarity and may be involved in regulation of the same programs as BCL6.

4.1.1 Choice of Technique

To examine protein-protein interactions on a large scale, two techniques are frequently used:

yeast two-hybrid and affinity purification. Yeast two-hybrid makes use of hybrid transcription

factor domains fused to proteins of interest, and measures the expression of reporter genes driven

by the association of those hybrids. Affinity purification is concerned with the associations

formed by tagged bait proteins with interactors, which are identified by mass spectrometry

following immunoprecipitation or pull-down. In comparison with affinity purification, yeast two-

hybrid is more likely to detect weak or transient interactions, or interactions between low

abundance proteins, because it makes use of a cDNA library, and is the method of choice when

focusing on proteins only expressed in rare or difficult-to-use cell types. However, this technique

has several limitations. By definition yeast two-hybrid takes place within yeast, in a different

cellular environment from mammalian proteins that are the target of the experiment. As such,

proteins and protein fragments may not maintain their ability to fold correctly and associate with

interactors in the absence of the conditions, associated proteins, and post-translational

modifications present in their normal cellular environment. Additionally, any incorrectly folded

or localized proteins may fail to interact with their usual partners but may instead form spurious

interactions, giving rise to false positives.

In contrast, the affinity purification approach is more versatile in the systems and cell lines used,

allowing interactions to occur in their native environment, but has a number of limitations as

well. In many cases, affinity purification has problems identifying true interactions, resulting in

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more false negatives. For instance, immunoprecipitation/pull-downs may not be able to detect

interactors that involve low-abundance proteins. The solubility of bait and prey proteins can also

generate false negatives, as conditions required to lyse cells and solubilize bait or prey proteins

may not be compatible with preserving protein-protein interactions. This limitation is extremely

relevant when considering weak and transient interactions, such as those involving transcription

factors, and thus making this limitation an obstacle in identifying protein-protein interactions

involving these proteins. Use of chemical cross-linking may be able to overcome this weakness,

however this introduces additional complications to the pull-down method and may cause

aggregation within the sample.

BioID, which covalently labels proximal proteins in living cells, is useful in overcoming some of

the limitations of yeast two-hybrid and affinity pull-downs. Unlike yeast two-hybrid, it detects

potential interactions in their normal cellular context and in comparison to affinity purification is

more capable of detecting weak interactions that may be lost due to solubility issues. Because

biotinylation is covalent and occurs before solubilisation, labelling of interactors is preserved

even through harsh lysis, and thus evidence of weak and transient interactions can be preserved.

BioID has its own set of limitations, however. Because it relies on the expression of exogenous

proteins fused to a BirA* tag, it is necessary that this tag not interfere with protein-protein

interactions or with assembly and folding of the bait protein. The fusion bait protein must display

the same attributes as wild-type or endogenous protein to identify true interactions and avoid

false positives. For the experiments I have conducted, the observation of BirA*-BCL6

interaction with NCOR, BCOR, and SMRT gives confidence in the other observed interactions.

Additionally, along with affinity purification, BioID shares the weakness of not identifying low-

abundance interactors that may not be present in sufficient amounts for identification following

purification and tandem mass spectrometry.

Further limitations arise concerning biotinylation of proximal interactors. One strength of

biotinylation is that it is a relatively rare post-translational modification in mammalian cells.

However, it does rely on the presence of primary amines in these nearby interactors. Covalent

attachment of biotin to lysines causes loss of charge and can affect post-translational

modifications, which may in turn affect the behaviour of these proteins. BioID is also dependent

on the ability to biotinylate nearby proteins, which is dependent on the number of primary

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amines available to be biotinylated; this means that the abundance of biotinylated proteins is not

necessarily indicative of the strength or abundance of association. Likewise, the lack of

biotinylation does not rule out interaction or proximity, and a lack of available primary amines in

interactors may result in false negatives. Additionally, the radius of biotinylation by BirA* is

estimated to be 10 nm, and proteins falling outside this radius may not be detected (Firat-Karalar

and Stearns, 2015). Related to this, the placement of the BirA* tag within the fusion protein can

be a particular concern: an N-terminal placement means that some proteins interacting with the

C-terminus of BCL6 or BCL6B may not fall within this radius, and therefore be missed as

interactors. Follow-up experiments may overcome this by making use of a C-terminal fusion tag

and comparing interactor datasets. Ultimately, in considering proteins biotinylated by BioID,

interactions can be placed into three categories: (i) direct interactions, either transient or stable,

(ii) indirect interactions connected by bridging proteins, or (iii) nearby proteins that do not

interact directly or indirectly. Validation on a case-by-case basis is necessary to determine which

proximal proteins fall into which category.

Modified HEK293 cells with a single Flp Recombination Target site incorporated into their

genome were chosen for these experiments. These cells are relatively easy to culture and

propagate, but the key advantage comes in the ability to readily generate stable cell lines

incorporating the gene for the BirA* fusion. This allows for the propagation of that gene within

the cell line and eliminates the need for subsequent transfections of the gene of interest.

Additionally, the FlpIn system used places the fusion gene under an inducible tetracycline

promoter within a single site in the genome. For the purposes of BioID, lower levels of

expression are preferred to higher levels, and near-endogenous levels of expression would be

ideal. Overproduction of BirA* fusion protein brings the possibility of mislocalization and

misfolding, as well as non-specific biotin labelling, thus increasing the number of potential false

positives. Expression of BirA*-BCL6 and BCL6B fusion proteins under an inducible promoter

and the fact that only one copy of the gene is integrated per cell gives us this control in the

experiment. However, HEK293 cells do not express BCL6 or BCL6B endogenously (Berglund

et al., 2008), which may call into question the biological relevance of some identified

interactions. For each interaction, biological relevance can be supported by looking at the

overlap of expression between bait and prey proteins in different contexts. More definitive

validation can be carried out in relevant cell lines, for instance BCL6-expressing lymphoma cell

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lines like Daudi or Ly1 cells (Berglund et al., 2008).

4.1.2 Identification of Statistically Significant Interactors

A significant number of non-specific background interactions are identified by BioID. It is

necessary to separate true interactors from contaminants. To do so, I turned to a probabilistic

algorithm, SAINT, that makes use of quantitative data in the comparison between bait-of-interest

and control datasets to assign probabilities of true and false interactions (Choi et al., 2012). To

obtain the most reliable information on true interactions, the number and nature of the controls is

crucially important, as these controls provide direct quantitative evidence for background

interactors. We used different BirA* control baits to mimic the experimental conditions of the

BCL6 and BCL6B experiments. Increasing the number of controls increases the robustness of

the SAINT analysis. Because SAINT uses quantitative data, in this case spectral counts of prey

proteins, low spectral counts in the bait datasets can result in the misidentification of some true

interactors as false negatives. Likewise, true interactors that have high spectral counts in control

data may be labelled as false interactors, also producing false negatives. The benefits of this

statistical analysis is that it removes many false positives.

4.1.3 Validation of Target Interactions:

Typically, high-throughput techniques produce large volumes of false positives and are also

associated with fairly high rates of false negatives. The validation of each individual interaction

may be difficult and time-consuming, and can be dependent on the chosen technique. The gold

standard for protein-protein interactions is co-immunoprecipitation, though as discussed above, it

has its limitations and is more suited to strong and stable interactions. Other techniques

commonly used include pull-downs, cross-linking followed by mass spectrometry or

electrophoresis, and label transfer interaction analysis. Interactions can be further validated by

quantitative and structural methods in defined systems with purified proteins. In vitro, the use of

analytical gel filtration can establish stoichiometry and complex size while detecting protein-

protein interactions, though it requires relatively large quantities of pure protein. For quantitative

measurement of binding constants, a wide array of techniques exist, from traditional methods

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such as isothermal titration calorimetry and surface plasmon resonance, to newer methods like

nanoscale thermophoresis and biolayer interferometry. These quantitative techniques are

extremely valuable in studying weak and transient interactions. Structural analysis in

complement to these quantitative techniques is one of the most information-rich methods of

characterizing interactions, giving a detailed view of complex formation and interaction modes.

X-ray crystallography, electron cryomicroscopy, and NMR are the most frequently used

methods, each having their own strengths and weaknesses.

All of the above techniques are low-throughput, however, and require large quantities of pure

protein, making them time and resource-intensive. They are most useful for further

characterization of an interaction once it has been validated. Bacterial expression systems have

traditionally been used to generate these large protein quantities, and such expression requires

the use of domains that are soluble and well-folded instead of full-length proteins. Experiments

done in vitro can lack bridging factors that facilitate the interaction, and tend to measure only

direct interactions. This can be a complication in validating interactors identified by BioID, as

many of these interactions may either be too weak to be reliably identified, or are proximal but

indirect interactors. Due to these intrinsic attributes, in vitro assays often fail to recapitulate high-

throughput interactions and lead to high rates of false negatives. In the validation of high-

throughput interactors, negative data from failed validations are rarely found in the literature,

which can lead to different groups repeating these failed validations. Despite this, and regardless

of the technique, the validation of interactions by independent groups increases confidence in the

verity of an interaction.

4.2 Interactions Identified by BioID

Many of the high-confidence putative interactors that I identified for BCL6 and BCL6B form

distinct protein complexes involved in a number of different critical biological functions. As is

expected given the function of both BCL6 and BCL6B as transcriptional repressors, many of the

identified interactors take part in transcriptional regulation, histone modification, and other

nuclear-localized processes. BCL6 is known to recruit co-regulators to carry out its repressor

activity, and among the putative interactors are members of a number of repressor complexes.

KDM1A (aka LSD1), NCOR/SMRT, BCOR, KDM2B, TLE1 and TLE3 are all corepressor

72

proteins that regulate transcription through histone modification (Farcas et al., 2012; Ghetu et al.,

2008; Jennings et al., 2006; Mottis et al., 2013; Wang et al., 2009). Additionally, BCL6 is known

to interact with other transcription factors (Miles et al., 2005; Phan et al., 2005b), and many of

the putative novel interactors are classified as DNA-binding transcription factors. There is

considerable overlap between the different interactor datasets for BCL6 and BCL6B with regards

to the different complexes identified.

For both BCL6 and BCL6B, several interactions are components of complexes associated with

HDAC3. Among these are members of the NCOR1/SMRT complex, including TBL1X,

TBL1XR1, and GPS2. Members of the BCOR-recruited PRC1-like complex, including BCOR,

PCGF, and KDM2B were also identified in both datasets. As has been previously described,

members of these complexes can be recruited directly through corepressor interaction with BCL6

to silence or repress genes that would otherwise interfere with the germinal centre reaction

(Ahmad et al., 2003; Ghetu et al., 2008; Hatzi et al., 2013). That they also appear as BCL6B

interactors may indicate that BCL6B is also capable of direct interaction with the corepressors

NCOR1, SMRT, and BCOR to recruit these complexes. Two members of the

Groucho/Transducin-Like Enhancer of split family, TLE1 and TLE3, as well as related

corepressor protein Amino terminal Enhancer of Split (AES) and transcription factor TCF7L1

were also identified as putative interactors for both proteins. These proteins regulate transcription

in a number of signalling pathways including Ras, Notch/Wnt, and Wingless by interacting with

transcription factors and recruiting HDAC proteins to modify chromatin (Arce et al., 2009). In

addition to this corepressor activity, TLE1 and TLE3 can directly interact with and modify

chromatin (Sekiya and Zaret, 2007).

Four members of the CTF/NF family of transcription factors, NFIA, NFIB, NFIC, and NFIX

were also identified. These transcription factors share between 60-70% sequence identity and

have been known to homo- and hetero-dimerize to carry out their biological functions

(Gronostajski, 2000). These functions are diverse and include roles in gliogenesis and

inflammation (NFIA), glioblastoma and brain development (NFIB), tooth development (NFIC),

and neurogenesis and hematopoiesis (NFIX) (Deneen et al., 2006; O’Connor et al., 2015; Steele-

Perkins et al., 2003, 2005).

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For BCL6B, members of the BRAF-histone deacetylase complex (BHC) including lysine

demethylase 1A(KDM1A), transcription factor ZMYM2, and GSE1 were also identified (Gocke

and Yu, 2008). This complex interacts with CoREST and the NuRD repressor complex to repress

neuron-specific genes in neuron differentiation (Sáez et al., 2015). KDM1A has also been

implicated in breast cancer metastasis through its interactions with the NuRD complex (Wang et

al., 2009).

Pre-mRNA splicing components were also identified as a cluster of closely related proteins

interacting with BCL6B. These components include splicing regulator slu7, RNA helicase

DHX40, and peptidyl isomerase PPIL3. The functions of these proteins are not well

characterized, but all are known to have a role in pre-mRNA processing (Urtasun et al., 2016; Xu

et al., 2002; Zhou et al., 2001).

Complexes containing core histone components were also identified as strong interactors for

BCL6 and BCL6B. These may be true interactions, given the histone-modifying nature of the

corepressor complexes recruited by the bait proteins, however it is also possible that they are

background contaminants or proximal proteins that do not interact directly with BCL6 and

BCL6B. Histone proteins are abundant proteins frequently identified in proteomic experiments

using HEK293 cells (Mellacheruvu et al., 2013). Likewise, the most likely explanation for the

observation of ribosomal subunit proteins such as RPS10, RPS18, and RPS19 is that they are

contaminants not filtered by SAINT in comparison with our controls. Indeed, comparison of

BCL6 and BCL6B datasets with controls in the CRAPome remove many of these proteins as

statistically significant interactors.

While there is considerable overlap between the interactors identified for BCL6 and BCL6B,

particularly with regards to the members of different repressor complexes, there were a large

number of interactors observed for BCL6B that were not observed for BCL6. Many of these

proteins are not associated with any of the other interactor found in the dataset, and many of

those are transcription factors. Though BCL6B protein is observed to be highly expressed in the

heart and lung, transcripts of BCL6B appear to be ubiquitously expressed at low levels in most

cells and tissues. This expression profile taken in tandem with BCL6B’s apparent interaction

with numerous different transcription factors may suggest that BCL6B is able to exert its effects

in different cell types by interacting with other cell-specific transcription factors. In this case

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BCL6B would recognize and bind its own specific sequence within DNA and then tether its

interacting TF partner to that region, or vice versa. One member of the transcription factor pair

would recognize and bind a given DNA sequence while the other partner would recruit its own

set of DNA- or histone-modifying enzymes to act upon the target gene (Figure 4.1).

Figure 4.1: Modes of BCL6B-mediated repression of target genes. A) BCL6 recruits a directly interacting

BCL6B-specific corepressor complex to repress its target gene. B) BCL6B tethers a partner interacting transcription

factor (TF) to the genome and the partner TF recruits its own specific corepressors to repress BCL6B target gene. C)

The situation is reversed and BCL6B is tethered to the genome by its TF partner, recruiting BCL6B-specific

corepressor to repress TF target genes.

This method of transcriptional regulation has previously been seen with other BTB-ZF protein

including BCL6. Interactions between the zinc finger region of BCL6 and transcription factors c-

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Jun, JunB, and JunD are integral to the repression of AP-1 genes by BCL6 (Vasanwala et al.,

2002). This method of transcription factor interaction to enhance protein function is also

observed among the GATA family of zinc finger transcription factors. Like BCL6B, members of

this family are highly expressed in certain tissues and ubiquitously expressed at lower levels.

They physically interact with more expression-restricted transcription factors to carry our tissue-

specific functions (Molkentin, 2000). GATA-3, for instance, interacts directly with TCF7L2 to

tether it to the GATA3 recognition sequence in breast cancer cells to repress genes involved in

breast cell differentiation (Frietze et al., 2012).

4.2.1 The BCL6 BTB domain interacts with TLE1 and TLE3

The interaction between BCL6 and both TLE1 and TLE3 was validated by co-

immunoprecipitation using full-length TLE1/3 and the BTB domain of BCL6. TLE1 and TLE3

are both ~83kDa corepressor proteins that share a conserved protein structure (Figure 4.2).

Figure 4.2: Schematic of conserved TLE1/3 domains. Domains are displayed from N-terminus to C-terminus

with their function annotated beneath them. Q-rich: Glutamine-rich region—mediates oligomerization, histone

binding, and HDAC interaction; GP: glycine-proline rich region; CcN: site of post-translational modification and

nuclear localization; SP: serine-proline rich region; WD Repeats: domain containing repeats of WD40 motif—

mediates interaction with transcription factors.

Overall they share 80% sequence identity, with an N-terminal glutamine-rich Q-region that has

86% identity and a C-terminal WD40 repeat region that has 95% identity. They function in many

different cellular pathways including neurogenesis, Notch/Wnt signalling, and regulation of

inflammation (Chen and Courey, 2000; Ramasamy et al., 2016). There is also considerable

overlap in the expression of TLE1, TLE3, and BCL6 (Su et al., 2004). To carry out their

repressional activity, TLE1 and 3 recruit HDAC through an N-terminal GP domain, but also

possess intrinsic histone-binding and chromatin-modifying ability(Chen et al., 1999; Sekiya and

Zaret, 2007). Crystal structures have been solved for both the WD40 and Q-regions of TLE1,

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both of which are known to bind different transcription factors to recruit TLE for repression

(Chodaparambil et al., 2014; Jennings et al., 2006). TLE1 and TLE3 are co-repressors for a

number of transcription factors that are shared between TLE1 and TLE3, among them HES1 and

Prop1, which are involved in neurogenesis. These transcription factors bind to the TLE1/3

WD40 domain by a conserved engrailed homology (eh1) domain (Carvalho et al., 2010). Other

transcription factors interact with the WD40 domain through a conserved WRPW motif, and the

crystal structure of TLE’s WD40 domain has also been solved in complex with peptides

representing the conserved motifs for both eh1 and WRPW (Jennings et al., 2006). Direct

interaction between the BCL6 BTB domain and TLE1/3 is likely to be mediated through either

the WD or Q domains. Nothing resembling the WRPW nor eh1 motifs are found in BCL6 or

BCL6B, and likewise, there is no sequence in either TLE1 or TLE3 resembling the BBDs from

NCOR/SMRT and BCOR. If the interaction between these proteins is direct, it will have to

involve a different interaction sequence, and may not occur through the lateral groove of BCL6.

The use of in vitro assays will be necessary to determine whether this is a direct physical

interaction. Otherwise, it may be necessary to determine if other proteins identified in this

experiment are acting as a scaffold for the interaction between BCL6 and TLE1 or TLE3.

4.2.2 The BCL6 BTB domain was not observed to interact with NFIA

Validation of the interaction between BCL6BTB and NFIA was also attempted by co-

immunoprecipitation, but results were negative. This may be a true result, and NFIA is not an

interactor with BCL6 but merely a BioID false positive proximal protein. Alternatively, NFIA

may be a direct interactor and the interaction was not observed by coIP because it is too weak,

was disrupted by protein solubilisation or wash steps, or interacts with a different region of

BCL6 than the BTB domain. NFIA could also be an indirect interactor, requiring other proteins

not present in sufficient quantities for the interaction to be detected.

4.3 BCL6B Interacts with the SMRT BBD

The interaction between BCL6B and the SMRT BBD was validated in vitro and using a

quantitative technique to determine binding constants. BTB domains from BCL6 and PLZF were

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also examined using this technique for comparison. The interaction was observed in vitro,

indicating that this is a direct interaction between BCL6B and SMRT and gives confidence that

the other BCL6B interactions with NCOR1 and BCOR are also true interactions based on their

known interaction with the BTB domain of BCL6 (Ahmad et al., 2003; Ghetu et al., 2008). It

was previously thought that BCL6B cannot regulate its transcriptional repressor activity without

the presence of BCL6 (Takenaga et al., 2003). The interaction of BCL6 with BCL6B was

necessary for BCL6B interaction with the corepressor Sin3A and HDAC3 in order to carry out

repression. Furthermore this interaction was thought to be mediated through heterodimerization

of the BCL6B and BCL6 domains. BCL6-BCL6B heterodimerization has yet to be replicated by

our lab (unpublished data), and the possibility exists that BCL6-BCL6B interaction may occur

through some other mode.

Because the interaction was validated in vitro, BCL6B can directly interact with corepressors

independent of the presence of BCL6. In comparing binding constants as determined by MST,

the interaction of BCL6 with SMRT (KD = 0.984μM) is approximately ten-fold stronger than that

of BCL6B (KD = 10.9μM). Dissociation constants measured for the BCL6BTB-SMRT BBD

interaction have ranged from 1.11μM (SPR) to 11.4μM (ITC). The constant measured for BCL6

by MST is fairly close to that of SPR, giving confidence in the MST results. It has previously

been found that the SMRT BBD does not bind the PLZF BTB domain (Ahmad et al., 2003),

which is consistent with our observations (data not shown).

The NCOR and BCOR corepressor fragments bind BCL6 in an extended conformation in the

lateral groove formed at the dimerization interface. Binding occurs only at the BTB domain, and

each BTB dimer binds two peptide fragments in a non-cooperative manner. Additionally, this

binding has not been found to induce a significant change in the BCL6 BTB main chain (Ahmad

et al., 2003; Ghetu et al., 2008). Thirty residues in the BCL6 BTB domain are buried upon

complex formation with SMRT; of these, nineteen residues are conserved in BCL6B, four are

represented by similar residues, and seven are different. Most of the different residues are found

within the β1 structural motif, and the effect of these differences is not known. One key

difference is observed with regards to arginine-24 in BCL6, which is a glutamate in BCL6B. The

side-chain of this arginine forms an ion pair with a glutamate at position 1427 in the SMRT

BBD, and the loss of this interaction may in part explain the difference in binding constants

between BCL6 and BCL6B. Structures of BCL6 in complex with either SMRT or BCOR BBD

78

peptides show that the side chain of R24 adopts a significantly different position compared to the

unliganded form of BCL6. It should also be noted that residues analogous to N21 and H116 in

BCL6 are conserved in BCL6B. Mutations to these residues in BCL6 are known to disrupt lateral

groove interaction with the BBD and their presence is required for binding.

4.4 Evolutionary Conservation of BCL6/B and the BBD

Phylogenetic analysis of the BTB domains based on their multiple sequence alignment reveals

that sequences cluster neatly into two groups based on their identity as BCL6 or BCL6B

proteins. Though a possible homolog to BCL6 is present in the tunicate, the identification of this

protein as similar to BCL6 is based upon high sequence identity in the ZF domains. Comparison

of the BTB domain of the tunicate BTB-ZF protein shows moderate sequence identity (~35%)

with a number of different human BTB-ZF proteins, including BCL6. Thus, whether or not this

is a true BCL6 homolog is difficult to determine. However, the protein sequence identified for

the Japanese lamprey bears more resemblance to the set of BCL6B proteins than to BCL6. From

this, it is assumed that the ancestral version of the BCL6/B gene bears closer sequence identity to

BCL6B than to BCL6. The emergence of a BCL6 gene roughly coincides with the evolution of

jawed vertebrates (gnathostomes) and the development of an immunoglobulin-based adaptive

immune system. The adaptive immune system is a recent evolutionary feature, unique to

vertebrates. Jawless fish (agnathans) are known to have a humoral immune response that differs

from that of gnathostomes, which uses leucine-rich repeat proteins to generate antigen receptor

diversity (Cooper and Alder, 2006).

From these data, it could be inferred that BCL6 arose from the ancestral BCL6B gene following

a gene duplication event, and that BCL6AB, found only in ray-finned fish, arose from a

subsequent duplication. Two whole genome duplications have been observed in the vertebrate

lineage, one of which occurred just after the divergence between jawed and jawless vertebrates

(Dehal and Boore, 2005) and this may be the cause of the duplication in BCL6/B. Following this

duplication, neofunctionalization of BCL6 or subfunctionalization of the role played by BCL6B

may have occurred in some lineages. Within some vertebrates, including fish and most

mammals, BCL6B was retained for its function, and the function of BCL6 diverged. In birds and

lizards, BCL6B and BCL6 function may have overlapped, leading to the loss of BCL6B, or it

79

may be that the biological role played by BCL6B was not essential to survival; this is supported

by knockout mouse models. The role of BCL6/B may not initially have been as a master

regulator in the immune system, however. Notably, germinal centres are a feature found only in

gnathostomes. Ancestral BCL6/B proteins may have played a different biological role, and been

co-opted for their current role in immune system development.

Likewise, NCOR1 and NCOR2 (SMRT) are likely products of gene duplication, with an

NCOR1-like protein being the ancestral form according to its presence in agnathans. Based on

knockout mouse models, NCOR1 and NCOR2 are non-redundant, and loss of either gene is fatal

(Mottis et al., 2013). Conservation of the BBD motif in all three corepressors indicates that this

sequence is important to corepressor function and also vital to the survival of the organism. The

BBD may be conserved in order to preserve the interaction with BCL6/B. This is borne out by

Shannon Entropy data, which shows that the lateral groove is very well conserved in BCL6 and

to a lesser extent in BCL6B. The relative lack of conservation in the BCL6B lateral groove may

indicate that there are other interactions besides NCOR1/SMRT and BCOR that involve binding

to the lateral groove in BCL6B. The difference in residues between the BCL6 and BCL6B lateral

grooves may represent a gain of function in the divergence between these two proteins, allowing

for the mediation of additional interactions. The lack of consensus between the NCOR1/SMRT

and BCOR BBDs supports the ability of the lateral groove to bind different sequences as part of

its function.

Speculation could be that it is important in other interactions that are also mediated in part by

residues in the lateral groove, and that this variation could be a gain-of-function mutation for

BCL6B or a vestige of an older function not involving the known BBDs. Given the difference

between NCOR1/2 and BCOR sequences, it is possible that a number of other peptides would be

able to interact with the lateral groove. Indeed, the most important feature of residues that form

these binding motifs is that they carry out van der Waals interactions that result in residues being

buried.

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4.5 Co-evolution of BCL6 and BCL6B Dimerization Residues

One important aspect of BCL6 biology is its ability to interact with BCL6B, possibly through

heterodimerization, thereby expanding their function by recruiting additional interactions. An

exploration of the covariance between inter-monomer residues that take part in BTB domain

dimerization was undertaken using the GREMLIN covariance webserver to evaluate this

possibility. In order to undertake statistically significant analysis of covariance, GREMLIN

makes use of several thousand BTB domain sequences from related proteins across many

species. As such, the determination of covariance between residues has implications for all BTB

domains that may form homo- and heterodimers, and not just BCL6-BCL6B.

Three pairs identified as co-varying were analogous between BCL6 and BCL6B, and a fourth

pair was identified with BCL6B as the seed sequence. All members of these pairs are at least

partially buried in the BCL6 BTB dimer. The pairs represent interactions between the α1 of one

monomer and α2 or α3 of the other, and take place at the “top” of the dimerization interface.

Only residues in these structural elements, and not the β1 and β5 sheets, which are also known to

be domain-swapped in dimerization, are the location of residues that co-evolve most strongly.

This may indicate that the α-helices that take part in dimerization are responsible for determining

the specificity of interaction between different BTB domains, and that residues at these positions

must be complementary for interaction to occur. This could be tested by systematically mutating

these residues and observing whether interactions are destabilized. Likewise, if these interactions

are important for specificity of dimerization, then they may be used to predict the

heterodimerization of different BTB proteins by identifying complementary residues at each

position in different BTB-ZF family members. Heterodimeric interactions between Miz1 and

BCL6, and Miz1 and Nacc1 have been solved structurally using tethered BTB domains (Stead &

Wright, 2014). The residues in Miz1 at these positions are nearly all identical to those in BCL6

and BCL6B. That they do have such co-variance among different BTB domains implies their

importance for the formation of BTB dimers.

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4.6 Expression and Purification of the BCL6B BTB domain

Further characterization of BCL6B in vitro will require the use of large quantities of pure BTB

domain, especially for crystallization. Expression and purification of mammalian proteins in

bacterial systems can often be difficult and result in the production of poorly soluble proteins

prone to aggregation. The use of recombinant protein tags can greatly contribute to the solubility

of the protein expressed, but difficulties can arise when these tags are removed. Such was the

case with the purification of BCL6B: following protease cleavage of a thioredoxin tag, cleaved

BCL6B BTB readily aggregated, resulting in poor yields of pure protein. Optimization of

solubilizing conditions using buffer screens failed to dramatically improve upon initial

purification conditions (data not shown), however, it was noted that LSZ Life Sciences uses

sarcosyl, an anionic detergent, in the storage buffer for BCL6B protein. The use of sarcosyl as an

additive improved the solubility of BCL6B more than 60-fold and increased the yield of pure

BCL6BBTB. Circular dichroism showed that in sarcosyl BCL6B has the characteristics of a

natively-folded BTB domain and BCL6B purified by size exclusion matches the expected profile

of the BCL6B BTB domain with no soluble aggregates present.

What role sarcosyl is playing in solubilizing BCL6B is not known. The final concentrations of

sarcosyl were 0.1-0.3% w/v, below the critical micelle concentration for sarcosyl of 0.7%, so

solubility is likely not being caused by integration into micelles, as is the case for solubilisation

of membrane proteins (Seddon et al., 2004). A likely explanation is that the hydrophobic tail of

sarcosyl is interacting with exposed hydrophobic regions of BCL6B and shielding them from the

aqueous environment, while leaving the charged-polar head exposed. In optimizing solubilizing

conditions it was found that lower concentrations of sarcosyl do lead to increasing levels of

aggregation of BCL6B.

Initial conditions for the purification of BCL6BBTB were adopted from previous successful

purifications of BCL6BTB. There may be several explanations as to why BCL6B is not soluble

under the same conditions as BCL6. One potential culprit could be the presence of helix-

breaking proline, P91 in the middle of α4, not present in BCL6. Mutation of the BCL6B protein

with a P91I substitution to mimic the residue found in BCL6 did not improve solubility,

however. Likewise, attempts to expand the C-terminus of the BTB domain did not improve

82

solubility, and it seems likely that the region encompassing residues 6 to 136 best represents the

BTB domain in BCL6B.

An additional issue with the purification of BCL6B was the off-target cleavage of the BTB

domain into two pieces of ~5 and 10kD respectively (data not shown) by thrombin following the

addition of sarcosyl. To overcome this, BCL6B was subcloned into a vector expressing a SUMO

tag fusion protein that can be cleaved with a SUMO protease (Malakhov et al., 2004). Making

this switch allowed us to maintain the same expression and purification strategy and eliminate

the thrombin digestion step. The ULP1 SUMO protease is active in the presence of sarcosyl, as is

thrombin protease from both bovine and human sources, which may be useful to note should

sarcosyl be used in the purification of other recombinant proteins.

The presence of sarcosyl has, unfortunately, been an obstacle to crystallization of BCL6B.

Approximately 800 different conditions have been tested for BCL6B crystallization by the vapor

diffusion method, producing a range of results from clear conditions to precipitation to

tetrahedral crystals that extinguished strongly. However, none of these conditions have produced

crystals that diffract well, and many of the larger crystals found in conditions have been sarcosyl

salt crystals. Sarcosyl readily crystallizes, and has resulted in false positives in many of the tested

crystallization conditions. Going forward, one solution to obtaining crystals would be to use an

entirely different expression system that may improve the folding and solubility of BCL6B,

eliminating the need to use sarcosyl for solubilization. The use of mammalian expression

systems, such as piggyBac, may be used to produce large amounts of BCL6B for expression

from mammalian cells for future work requiring pure protein.

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5 Conclusion and Future Directions

5.1 Conclusions

BCL6 plays a role in many different cellular processes, most of which concern hematopoiesis

and immune system development. Its most well-characterized role is as a master regulator of the

germinal centre reaction for B cell development in the humoral immune response. The roles

BCL6 plays in other processes, including TFH development, differentiation and inflammation in

TH2 cells, and T cell activation are much less well-characterized. Additionally, there is evidence

that BCL6 interacts with an ortholog, called BCL6B, with which it has high sequence identity

and which has been implicated in some of these same processes. To date, the function of BCL6B

in human biology has not been well-studied. To increase our understanding of BCL6 and

BCL6B, we set out to expand the known protein-protein interactions for each of these two

proteins using BioID, a proximity dependent-labelling method.

BioID identified 24 novel interactions for BCL6 and 75 novel interactions for BCL6B. The

proximal labelling nature allows for the identification of indirect interactors that form part of

interacting complexes. Among the interactors identified by BioID, a number of functional

complexes emerge. For both BCL6 and BCL6B, members of the NCOR/SMRT and BCOR

complexes were identified, as well as members of the Groucho/TLE corepressor and CTF/NFI

family, which would constitute novel interacting complexes.

Furthermore, interactions between the BCL6 BTB domain and the TLE1 and 3 corepressors were

validated by co-immunoprecipitation. The interaction between the BCL6B BTB domain and the

BTB-domain binding region of the SMRT corepressor was also validated and the binding

strength of this interaction was quantified in vitro.

Due to the overlap in interactors between BCL6 and BCL6B and their conserved sequence

identity, inquiries into the evolutionary history of the two proteins was undertaken. It was found

that their appearance coincides with the evolution of vertebrates and the adaptive immune

system, and the BCL6B, though it more closely resembles the ancestral protein, is not as well-

conserved in jawed vertebrates as BCL6. Additionally, the lateral groove, which mediates

important protein-protein interactions with the corepressors NCOR1/SMRT and BCOR, is more

84

well-conserved in BCL6. Finally, sequence analysis of the BTB domain was undertaken using

co-variance to identify residues important to BTB dimerization. These results may provide a

springboard for future research to elucidate the novel biological roles of BCL6 and BCL6B. In

particular, the direct interaction between BCL6B and the NCOR1 and SMRT corepressors could

be further characterized. Additionally, validation of the interaction between BCL6 and TLE1/3

opens up an avenue for future exploration with potential implications in Notch/Wnt signalling or

inflammation.

5.2 Further Characterization of BCL6-TLE interaction

Having validated the interaction between the BCL6 BTB domain and TLE1 and TLE3,

determining if this is a direct interaction would be the first step towards further characterization.

Co-localization of endogenous proteins in relevant cell lines where there is overlap of

expression, for instance TFH (T cell) or Ly1 (DLBCL) cell lines will be important for establishing

biological relevance. A method such as glutathione-S-transferase pull-down could be employed

to determine direct interaction, and would be followed by domain mapping of the TLE proteins

to determine the regions involved in this interaction. Further investigations into the biological

relevance of the interaction could be carried out by RNAi knockdown assays or transgenic

mouse models. Luciferase reporter assays could be used to determine the effect of TLE-BCL6

interaction on gene repression and chromatin immunoprecipitation could be used to identify

target genes bound by the BCL6-TLE complex. Additionally, quantitative PCR could be used to

determine which genes have their transcription levels affected by TLE-BCL6 interaction. Having

established a direct interaction and the biological implications of this interaction, quantification

of BCL6-TLE binding could be carried out using purified proteins expressed in an E. coli

expression system. These purified proteins could also be used in the crystallization and structural

analysis of a BCL6BTB-TLE complex.

85

5.3 Mapping of Interactions with Repression Domain Mutants

The BTB domain and RD2 regions are well characterized for their ability to mediate protein-

protein interactions. The most well-characterized interaction region of the BTB occurs through

the lateral groove, and this interaction can be disrupted through the mutation of key residues

(Ahmad et al., 2003). Binding to the RD2 region can also be abrogated by acetylation of key

lysine residues, or mutations mimicking acetylation (Huang et al., 2014). Different sets of

constructs with the relevant disrupting mutations could be used in BioID to identify which

interactions are mediated by each domain by comparison the datasets from these specific mutants

with the datasets from wild-type BCL6 and observing which interactions are lost.

5.4 Structural Characterization of BCL6B BTB Domain and Interaction with SMRT

A number of strategies may be pursued towards obtaining the crystal structure of the BCL6B

BTB domain. Continued exploration of the crystallization space may yield protein crystals that

give good diffraction data. In this case, increasing the concentration of pure protein used in

crystallization trials may be the most effective solution. Failing this, purification may need to be

optimized to obtain soluble BCL6B without the use of sarcosyl. This could be done through

further optimization of the protein buffer or the use of orthologs that may be more suitable

candidates for purification and crystallization. Alternately, it may be that an additive or co-factor

would help with purification. The inclusion of SMRT peptide in the purification process may

both improve solubility and allow for the crystallization of the BBD-BTB interaction between

BCL6B and SMRT.

Another alternative is to express BCL6B in a mammalian system such as the PiggyBac system

(Akhtar et al., 2015), which has been optimized to express high levels of recombinant protein in

HEK293 cells. Such expression may allow for proper folding and modification lacking in the E.

coli systems currently used, while still providing an appropriately large amount of protein for

crystallization and other assays.

86

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Appendices

Table A1: BCL6/B Proteins included in bioinformatic analyses. Table includes source

organism, label, accession number, and length in amino acids.

Sequence Organism Accession No. Length

_Hs_BCL6 Homo sapiens NP_001124317.1 706

_Mm_BCL6 Mus musculus NP_033874.1 707

_Gg_BCL6 Gallus gallus NP_001012948.1 708

_Tg_BCL6 Taeniopygia guttata XP_002191364.1 709

_Cp_BCL6 Chrysemys picta bellii XP_005308112.1 709

_Ac_BCL6 Anolis carolinensis XP_003229443.2 708

_Xt_BCL6 Xenopus tropicalis NP_001116278.1 702

_Dr_BCL6 Danio rerio NP_957028.1 704

_Tr_BCL6 Takifugu rubripes NP_001072069.1 703

_Cm_BCL6 Callorhincus milii XP_007883672.1 671

_Lc_BCL6 Latimeria chalumnae XP_005992513.1 721

_Ci_BCL6 Ciona intestinalis NP_001071662.1 825

_Hs_BCL6B Homo sapiens NP_862827.1 480

_Mm_BCL6B Mus musculus NP_031554.1 474

_Cp_BCL6B Chrysemys picta bellii XP_008174793.1 454

_Dr_BCL6B Danio rerio XP_001333002.1 550

_Tr_BCL6B Takifugu rubripes XP_003966968.1 534

_Lc_BCL6B Latimeria chalumnae XP_014346158.1 666

_Lj_BCL6B Lethenteron japonicum N/A 841

_Dr_BCL6AB Danio rerio NP_001093544.1 565

_Tr_BCL6AB Takifugu rubripes XP_003975829.1 638

103

Figure A1: Unrooted phylogenetic tree of the BTB domains. The tree was generated using

BTB domain sequences of the BCL6 and BCL6B proteins from representative organisms.

Proteins that cluster with BCL6 and with BCL6B sequences were noted as such. Sequences were

aligned using MUSCLE and the tree was generated by FigTree (v 1.4).

104

Figure A2: Unrooted phylogenetic tree of the full-length BCL6/B proteins. The tree was

generated using full sequences of the BCL6 and BCL6B proteins from representative organisms.

Proteins that cluster with BCL6 and with BCL6B sequences were noted as such. Sequences were

aligned using MUSCLE and the tree was generated by FigTree (v 1.4).

105

Figure A3: Unrooted phylogenetic tree of the ZF regions. The tree was generated using ZF

region sequences of the BCL6 and BCL6B proteins from representative organisms. Proteins that

cluster with BCL6 and with BCL6B sequences were noted as such. Sequences were aligned

using MUSCLE and the tree was generated by FigTree (v 1.4).

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Table A2: Summary of NCOR1/SMRT repressor complex members and PRC-like complex

members identified in representative organisms. Protein columns are coloured based on the

complex they are part of, with yellow showing NCOR1/SMRT complex members and green

showing PRC-like complex members. BCL6 and BCL6B proteins are included in blue for

comparison. Whether the BBD motif is present in the corepressor of each organism is also noted.

characterizing novel interactions of transcriptional ... novel interactions of transcriptional...

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