immunity supplemental information the foxo1 transcription ...€¦ · intrachromosomal ramos...

Immunity

Supplemental Information

The FOXO1 Transcription Factor Instructs

the Germinal Center Dark Zone Program

David Dominguez-Sola, Jennifer Kung, Antony B. Holmes, Victoria A. Wells, Tongwei

Mo, Katia Basso, and Riccardo Dalla-Favera

Figure S1, related to Figure 2: FOXO1 deletion alters the dynamics and polarity of GCs, but not GC formation. (A) Representative immunofluorescence analyses of spleens from Foxo1+/+x Cγ1-Cre (WT) and Foxo1L/LxCγ1-Cre (KO) mice at the indicated time points after SRBC immunization. Bcl6+ B cell clusters (early GC) are highlighted by white contours and arrows (day 4). Scale bar, 50 microns. (B) Dynamics of Bcl6 expression in WT and FOXO1 KO GC B cells. Flow cytometry analysis after intracellular staining. Plots representative of 3 animals per time point. (C) Bcl6 mRNA levels (quantitative RT-PCR), 8 days post-SRBC immunization (n=3)(average ± standard deviation). (D) Analysis of GC polarity (LZ/DZ) during the GC reaction, at the indicated time points post-SRBC immunization. GC B cells were gated based on Cd95 and Bcl6 expression (Bcl6hi, Cd95hi). Dot plots are representative of 4 animals per genotype. (E) Quantification of DZ/LZ ratios, as in (D) (average±standard deviation; n=4). (F) Analysis of the GC reaction (Left: bulk GC; Right: DZ vs LZ distribution), 3 weeks after SRBC immunization. Shown are representative flow cytometry dot plots for both WT and KO mice cohorts (n=3). The complete data and statistical analyses ( Student’s T-test, two-tailed, unequal variance; NS=p>0.05) are graphed in (G). (H) Percentage of plasma cells in spleen, 3 weeks after SRBC immunization. Shown are 2 representative plots. Complete data set graphed in (I) (n=3)!

A! B! C! F! H!

D! E! G! I!

Figure S2, related to Fig. 2. Analysis of gene expression profiles in Foxo1-null GC B cells. (A) Hierarchical clustering of the gene expression profiles of Foxo1+/+-Cγ1Cre (WT), Foxo1+/L-Cγ1Cre (WT), and Foxo1L/L-Cγ1Cre (KO) mice GC B cells using genes that are differentially expressed between WT and KO. (B) A previously reported gene signature discriminating DZ from LZ GC B cells both in human and mouse (Victora et al., Blood 2012) was used to interrogate the expression profiles of Foxo1+/+-Cγ1Cre (WT) and Foxo1L/L-Cγ1Cre (KO) mice GC B cells. The heat maps display the genes that are differentially expressed in WT vs KO samples (Student t-test, p<0.05) and those that are (C) up-regulated or (D) down-regulated in DZ versus LZ GC B cells, but are not differentially expressed upon Foxo1 deletion (See also Table S1).!

A! C! D!B!

WT!

KO!

WT!

KO!

Figure S3, related to Figure 3: Analysis of GC responses to NP-KLH immunization in Foxo1+/+-Cγ1Cre (WT) and Foxo1L/L-Cγ1Cre (KO) mice. (A) Representative plots of bulk GC populations (left panels) and DZ vs LZ distribution (right panels), 14 days after NP-KLH immunization (i.p). The graphs below show the complete dataset (average±standard deviation; Student’s T-test, two-tailed, unequal variance). (B) Analysis of the NP-affinity response in the same animal cohorts. Representative plots are shown in the top panels (dot/contour plots), while the complete analysis of gated populations is graphed below (WT n=2; KO n=3; average±standard deviation; Student’s T-test, two-tailed, unequal variance). (C) Analysis of the early GC response to NP-KLH (day 4 post-immunization). Representative plots of: GC gate (left panel), FOXO1 gate (intracellular staining; middle panel), Ig lambda surface expression (right panel) in early GC B cells. (D) Quantification of the results illustrated in (c) (n=3; average±standard deviation; Student’s T-test, two-tailed, unequal variance). Black, WT littermates; Red, Foxo1 KO littermates (KO+, non-recombined cells; KO-, Foxo1-null cells). !

A! B!

C ! D!

0!0.2!0.4!0.6!0.8!

1!1.2!1.4!1.6!1.8!

2!

WT! KO!

mAi

cda

mRN

A le

vels

(rela

tive)!WT !

KO !

AID DAPI !

a! b!

n=3!

Dominguez-Sola et al_Supplementary Figure S2!

Figure S4, related to Figure 3: Normal AID mRNA and protein levels in Foxo1-null GCs. (A) Immunofluorescence analysis for AID protein levels in Foxo1+/+;Cγ1Cre (WT) and Foxo1L/L;Cγ1Cre (KO) GCs from murine spleens. Scale bar, 100 micron. (B) Quantitative RT-PCR for Aicda transcript amounts in sorted mouse GC B cells (average ± standard deviation, n=3). Shown are the results of a representative analysis with one out of 3 different primer sets used, all producing equivalent results. Data are displayed as fold change over the expression of Aicda in WT GC B cells (set arbitrarily to 1), after normalization to the expression of a housekeeping gene (Actb).!!

A! B!

mAicda

mRN

A am

ount

s (re

lativ

e)!

C!

0!

5!

10!

15!

20!

25!

30!

35!

40!

45!

IgG1! IgG3! IgG1! IgG3!

Foxo1 +/+! Foxo1 f/f !

Perc

ent o

f sw

tiche

d ce

lls!

PBS! TAT-Cre!

B!

B220!

IgG

1!

A!PBS! TAT-Cre!

Foxo1!

Even

ts!

D!

Dominguez-Sola et al_Figure S5!

0!

0.2!

0.4!

0.6!

0.8!

1!

1.2!

1.4!

1.6!

IgG1_GLT ! ! IgG1_PST! mAID !Re

lativ

e m

RNA

amou

nts!

!

PBS! TAT-Cre!

Foxo1+/+! Foxo1L/L!Foxo1L/L!

Sample NameExvivo CSR_541_none_011.fcs Exvivo CSR_541_TAT-Cre_012.fcs Foxo1L/L!

Foxo1+/+! Foxo1L/L!

Figure S5, related to Figure 4: Defective IgG1 class switch recombination in Foxo1-null B cells upon in vitro activation.(A) Intracellular levels of Foxo1 protein in mock-treated (PBS) or Foxo1L/L (upon TAT-CRE dependent Cre-lox recombination) splenic B cells, activated with LPS+IL4 in vitro, assessed by intracellular staining and flow cytometry analysis, 3 1/2 days after stimulation. (B) IgG1 surface expression (as determined by flow cytometry), on the same samples shown in (A), representative of 3 independent biological replicates. (C) Percentage of IgG1 switched activated B cells, as assessed by surface staining and flow cytometry analysis (mean ± standard error; n=3). (D) IgG1 germline (GLT), post-switch (PST) and Aicda transcript levels at the experimental end-point (3 ½ days post-activation), as measured by quantitative RT-PCR (average standard ± deviation; n=3). Data are represented as fold change over the transcript levels in WT B cells (set arbitrarily to 1), after normalization to the expression levels of a reference housekeeping gene (Actb). "

Figure S6, related to Figure 4: Changes in the IgH locus transcriptional landscape and class-switch recombination upon Foxo1 deletion in mouse GC B cells. (A) Quantitative RT-PCR analysis of I-mu locus and of (B) different Ig class transcripts (germline, GLT and post-switch, PST) transcripts in mouse splenic Foxo1+/+ (WT), Foxo1L/L (KO) GC B cells and in Naive, non-GC B cells (Na), 14 days after immunization with SRBC (average±standard deviation, n=3). (C) Levels of surface+intracellular Ig isotypes in murine splenic GC B cells in the sample samples as in (A, B). (D) UCSC genome browser tracks showing the distribution of FOXO1 bound regions at the Ig locus (ChIP-Seq) in human CD77+ GC B cells (‘centroblasts’). (FOXO1 top, H3K27Ac, bottom; see Table S2). Below, distribution of mapped superenhancers and intrachromosomal 3D interactions (ChIA-PET) in Ramos B cells activated in vitro with CD40+IL4 (GEO record number GSE62063; Kieffer-Kwon et al, Cell 2013). !

c!

Dominguez-Sola et al_Supplementary Figure S6!

0!2!4!6!8!

10!12!

KO! WT! Na! KO! WT! Na! KO! WT ! Na ! KO! WT ! Na !Imu_exon ! Imu_Ste! Mu0_exon ! Mu0_Ste!

I-Mu sterile (ste) transcripts !

Rela

tive

mRN

A am

ount

s!

A!

IgG2b, G3, A and E transcripts!

Rela

tive

mRN

A am

ount

s!

B!

ramos_H3K27ac_SuperEnh

CB4_FOXO1_RDF

CB4_H3K27Ac_RDF


IgMap custom track

Intrachromosomal RAMOS Chia-PET connections supported by 2 or more independent PETs. Score: cap FDR at 10^-20, map -10*log10(FDR) to integers between 0 and 1000

Vega Protein-Coding Annotations

UCSC Genes (RefSeq, GenBank, CCDS, Rfam, tRNAs & Comparative Genomics)

Ca2Sa2

CeSe

Ie

Cg4Sg4

Ig4

Cg2Sg2

Ig2

Ca1Sa1

Cg1Sg1

Ig1

Cg3Sg3

Ig3

CdCmu

Smu

Imu

2[1.13165e-10]13[9.23962e-38]

2[1.90334e-10]

2[1.17971e-11]2[3.15771e-05]

2[5.89682e-09]2[2.07792e-05]

3[3.40087e-09]2[1.01135e-05]

2[6.7686e-07]

72[0]

2[1.94505e-07]13[0]

IGHA2AL928742.12AL928742.12AL928742.12

AL928742.12IGHE

RP11-731F5.1RP11-731F5.1

IGHG4 IGHG2RP11-731F5.2

RP11-731F5.2

AL928768.3IGHA1

IGHG1IGHG1IGHG1

IGHG3 IGHDIGHM

IGHJ6

IGHJ5IGHJ4IGHJ3IGHJ2IGHJ1

IGHD7-27

IGHD1-26

IGHD6-25

IGHD5-24

IGHD4-23IGHD3-22

abPartsIGH@

DKFZp686O16217epsilon IgE

immunoglobulin epsilon chain constant...IGHEIGHE

AY748447DKFZp686O16217

ELK2AP

ELK2APIg alpha 1-[alpha]2m

epsilon IgE IGHG1

IGHCgamma1FLJ00385FLJ00385

IGHDFLJ00382FLJ00382

IGHDFLJ00382

AK128652

CB4_FOXO1_RK059 chr14:105982646-106383590

1.97132 _

0 _

CB4_H3K27Ac_RK043 chr14:105982646-106383590

23.0434 _

0 _

FOXO1_Human GC B cells_GSE68349!

H3K27Ac_Human GC B cells_GSE68349!


CB4_FOXO1_RDF

CB4_H3K27Ac_RDF


IgMap custom track




Ca2Sa2

CeSe

Ie

Cg4Sg4

Ig4

Cg2Sg2

Ig2

Ca1Sa1

Cg1Sg1

Ig1

Cg3Sg3

Ig3

CdCmu

Smu

Imu

2[1.13165e-10]13[9.23962e-38]

2[1.90334e-10]

2[1.17971e-11]2[3.15771e-05]

2[5.89682e-09]2[2.07792e-05]

3[3.40087e-09]2[1.01135e-05]

2[6.7686e-07]

72[0]

2[1.94505e-07]13[0]

IGHA2AL928742.12AL928742.12AL928742.12

AL928742.12IGHE

RP11-731F5.1RP11-731F5.1


RP11-731F5.2

AL928768.3IGHA1

IGHG1IGHG1IGHG1

IGHG3 IGHDIGHM

IGHJ6


IGHD7-27

IGHD1-26

IGHD6-25

IGHD5-24

IGHD4-23IGHD3-22

abPartsIGH@



AY748447DKFZp686O16217

ELK2AP


epsilon IgE IGHG1



IGHDFLJ00382

AK128652

CB4_FOXO1_RK059 chr14:105982646-106383590

1.97132 _

0 _

CB4_H3K27Ac_RK043 chr14:105982646-106383590

23.0434 _

0 _Ramos_H3K27Ac_Super Enhancers (GSE62063)!


CB4_FOXO1_RDF

CB4_H3K27Ac_RDF


IgMap custom track




Ca2Sa2

CeSe

Ie

Cg4Sg4

Ig4

Cg2Sg2

Ig2

Ca1Sa1

Cg1Sg1

Ig1

Cg3Sg3

Ig3

CdCmu

Smu

Imu

2[1.13165e-10]13[9.23962e-38]

2[1.90334e-10]

2[1.17971e-11]2[3.15771e-05]

2[5.89682e-09]2[2.07792e-05]

3[3.40087e-09]2[1.01135e-05]

2[6.7686e-07]

72[0]

2[1.94505e-07]13[0]

IGHA2AL928742.12AL928742.12AL928742.12

AL928742.12IGHE

RP11-731F5.1RP11-731F5.1


RP11-731F5.2

AL928768.3IGHA1

IGHG1IGHG1IGHG1

IGHG3 IGHDIGHM

IGHJ6


IGHD7-27

IGHD1-26

IGHD6-25

IGHD5-24

IGHD4-23IGHD3-22

abPartsIGH@



AY748447DKFZp686O16217

ELK2AP


epsilon IgE IGHG1



IGHDFLJ00382

AK128652

CB4_FOXO1_RK059 chr14:105982646-106383590

1.97132 _

0 _

CB4_H3K27Ac_RK043 chr14:105982646-106383590

23.0434 _

0 _

Human chr14:105,854,694-106,527,362 (hg19)!


CB4_FOXO1_RDF

CB4_H3K27Ac_RDF


IgMap custom track




Ca2Sa2

CeSe

Ie

Cg4Sg4

Ig4

Cg2Sg2

Ig2

Ca1Sa1

Cg1Sg1

Ig1

Cg3Sg3

Ig3

CdCmu

Smu

Imu

2[1.13165e-10]13[9.23962e-38]

2[1.90334e-10]

2[1.17971e-11]2[3.15771e-05]

2[5.89682e-09]2[2.07792e-05]

3[3.40087e-09]2[1.01135e-05]

2[6.7686e-07]

72[0]

2[1.94505e-07]13[0]

IGHA2AL928742.12AL928742.12AL928742.12

AL928742.12IGHE

RP11-731F5.1RP11-731F5.1


RP11-731F5.2

AL928768.3IGHA1

IGHG1IGHG1IGHG1

IGHG3 IGHDIGHM

IGHJ6


IGHD7-27

IGHD1-26

IGHD6-25

IGHD5-24

IGHD4-23IGHD3-22

abPartsIGH@



AY748447DKFZp686O16217

ELK2AP


epsilon IgE IGHG1



IGHDFLJ00382

AK128652

CB4_FOXO1_RK059 chr14:105982646-106383590

1.97132 _

0 _

CB4_H3K27Ac_RK043 chr14:105982646-106383590

23.0434 _

0 _

Immunoglobulin Segments!

Intrachromosomal interactions, ChIA-PET, Ramos cells (GSE62063)!

Vega Protein-Coding Annotations!

IgG2a IgG2b IgA IgE0

5

10

15

Isotype

C!

D!

Surface & Intracellular protein expression!

0!

0.5!

1!

1.5!

2!

2.5!

3!

3.5!

KO! WT! Na! KO! WT! Na! KO! WT! KO! WT ! KO! WT ! Na! KO! WT ! Na! KO! WT ! Na!GLT! PST! GLT! PST! GLT! GLT_a! PST!

IgG2b! IgA! IgE! IgG3!

Table S5. List of antibodies used for flow cytometry (FC), immunofluorescence on paraffin embedded tissues (IF-P) and immunoblot (WB) analysis.

Molecule

Application

Species

Fluorochrome

Clone

Manufacturer

Anti-mouse antibodies B220 FC Mouse PCP RA3-6B2 BD PNA FC Mouse Biotin FL-1071 Vector PNA FC Mouse FITC FL-1071 Vector CD38 FC Mouse PE 90/CD38 BD Pharmingen CD95 FC Mouse PE Jo2 BD CD95 FC Mouse PE-Cy7 Jo2 BD CD86 FC Mouse APC GL1 eBioscience

CXCR4 Igλ

Igλ1,2,3

FC FC FC

Mouse Mouse Mouse

eFluor 450 PE, Biotin

Biotin

2B11 RML-42 R26-46

eBioscience Biolegend

BD Biosciences IgM FC Mouse Biotin R6-60.2 Southern Biotech IgM FC Mouse APC II/41 BD Pharmingen IgG1 FC Mouse Biotin 10.9 BD Pharmingen IgG1 FC Mouse APC X56 BD Pharmingen

IgG2a FC Mouse Biotin H106.771 Southern Biotech IgG2b FC Mouse Biotin LO-MG2b Southern Biotech IgG3 FC Mouse Biotin LO-MG3 Southern Biotech IgE FC Mouse Biotin 23G3 Southern Biotech IgA FC Mouse Biotin 11-44-2 Southern Biotech

BCL6 FC Mouse PE K112-91 BD FOXO1 FC/IF-P Mouse/Human Purified C29H4 Cell Signaling

AID CD21

IF-P IF-P

Mouse/Human Mouse/Human

Purified Purified

mAID-2 ab75985

eBioscience Abcam

Anti-human antibodies

IgD FC Human FITC IA6-2 BD CD3 FC Human FITC IM1281 Immunotech

CD38 FC Human PE HIT2 BD CD83 FC Human Biotin HB15e Biolegend

CXCR4 FC Human PE-Cy7 12G5 Biolegend FOXO1 FC/WB/IF-P Human/Mouse Purified C29H4 Cell Signaling

BCL6 IF-P/WB/ChIP Human/Mouse Purified N3 Santa Cruz Biotechnology

FOXO1 ChIP Human/Mouse Purified #ab39670 Abcam H3K27Ac ChIP Human/other Purified #39133 ActiveMotif

AKT WB Human/Mouse Purified C67E7 Cell Signaling p-AKT (S473) WB Human/Mouse Purified D9E Cell Signaling

Actin-B WB Human/Mouse Purified #A5441 Sigma CD23 IF-P Human Purified 1B12 Novocastra AID IF-P Human/Mouse Purified mAID-2 eBioscience

Secondary Reagents

Streptavidin FC/IF-P - eFluor450 - eBioscience Streptavidin FC/IF-P - Alexa350 Molecular Probes

Zenon IgG(H+L)

FC/IF-P FC

Rabbit Rabbit

Biotin/A647 Alexa546

- -

Life Technologies Life Technologies

IgG(H+L) IF-P Mouse Cy3 Jackson Immunoresearch

IgG (H+L) IF-P Rat Biotin #6430-08 Southern Biotech

IgG (H+L) IF-P Rabbit Envision Polymer Dako

Tyramide-FITC IF-P - FITC Perkin Elmer IgG (H+L) WB Rabbit HRP GE Healthcare IgG (H+L) WB Mouse HRP GE Healthcare

Supplemental Experimental Procedures Human tissue samples Tonsils were obtained from routine tonsillectomies at the Children's Hospital of Columbia-Presbyterian Medical Center. All samples were exempt from the requirement for informed consent as they corresponded to residual material obtained after diagnosis, from anonymous donors without identification of samples, in compliance with Regulatory Guideline 45 CFR 46.101 (b)(4) for Exempt Human Research Subjects of the US Department of Health and Human Services and with protocols approved by the Institutional Ethics Committee. Immunization protocols and treatment For the generation of GC responses, ~10-12 wk old mice were immunized by intraperitoneal injection of (1x109) SRBCs (Cocalico Biologicals) or 100 ug of NP (4-hydroxy-3-nitrophenyl-acetyl) conjugated to KLH (BioSearch) (in complete Freund’s Adjuvant); and generally analyzed at days 10–14 (or for specific experiments, at 4-6-8 days (Fig.S2, Fig. S3), or 3 weeks post-immunization (Fig. S1). To generate larger amounts of GC B cells (i.e. for gene expression profiling), we did two sequential i.p. SRBC injections (day 0, 1-2 × 108; day 5, 1 × 109) and collected cells at day 12. This protocol yielded about three-four fold more GC B cells (~12-15% of the B cell fraction) than with a single immunization. B cell isolation, staining, flow cytometry analysis and cell sorting. Mononuclear cell pools were isolated from mouse spleens at the indicated times post-immunization. Cell were separated by crushing the spleens through a 40 micron mesh in ice-cold PBS- 0.5%BSA -2mM EDTA +2% FCS. In some experiments (ie. cell sorting), we further enriched for B cells using negative selection with magnetic beads (B cell isolation kit, Miltenyi), following manufacturer’s instructions. Cell stainings were performed on ice in PBS +0.5% BSA (or PBS+2% Fetal Calf Serum) + 2mM EDTA. Details on antibodies are provided in Table S5. We included a prior step of incubation with Fc-block Reagent (Trustain FcX, Biolegend). GC B cells were identified within the B cell fraction (B220hi) as GL7hi (or PNAhi), CD95hi. Both Cxcr4 and Cd86 were used to distinguish DZ (Cxcr4hi, Cd86lo) and LZ (Cxcr4lo, Cd86hi) GC B cells, as previously reported (Victora et al., 2012; Victora et al., 2010). Plasma cells were identified as in a previous report (Bannard et al., 2013), by gating on Cd138hi, B220int cells within the GL7-, IgD-, Cd3- pool. For the detection of intracellular proteins (i.e. FOXO1 or BCL6), cell suspensions were fixed and permeabilized using the Cytofix-Cytoperm and Cytoperm plus buffers (BD Biosciences) and subsequently stained for 60 minutes at RT with specific antibodies. The anti-FOXO1 antibody was pre-labeled using the Zenon-Biotin or Zenon-Alexa647 kits (Life Technologies), or alternatively, with an anti-rabbit antibody conjugated Alexa 546 (Molecular Probes/Life Technologies). BCL6 was detected with a phycoerythrin-conjugated anti-BCL6 antibody. To generate cell cycle profiles, we used DAPI staining after permeabilization, and data was collected at 400 events per second. All flow cytometry analyses and cell-sorting procedures were done at the Herbert Irving Comprehensive Cancer Center Flow Cytometry Facility (Columbia University), using BD LSR-Fortessa or BD LSRII cell analyzers, and a BD FACSAria II sorter running BD FACSDiva software (BD Biosciences). FlowJo software (v 9.8.1 and v.10; TreeStar) was used for data analyses and plot rendering. Immunofluorescence analysis of paraffin-embedded lymphoid tissues For immunofluorescence microscopy analyses we used 3-μm-thick sections of formalin-fixed, paraffin-embedded tissues, as previously described (Cattoretti, 2013; Cattoretti et al., 2006). Mouse lymphoid tissues had been fixed overnight in 10% buffered formalin, post-fixed in 70% ethanol before dehydration and paraffin embedding. Heat-induced epitope retrieval was performed in citrate buffer (pH 6.0, + 0.5% Tween). Endogenous peroxidase was blocked in PBS plus 3% H2O2 and endogenous biotin using the Avidin-Biotin Blocking Kit (Vector), when required. All sections were further blocked in PBS containing 0.1%Tween, 3%BSA and 5% goat serum

(Jackson Immunoresearch), followed by incubation overnight at 4°C with specific primary antibodies (Table S5). After repeated washes in 0.1% Tween in PBS, sections were incubated for 1 h at room temperature with fluorochrome-, horseradish peroxidase- or biotin-conjugated antibodies and isotype-specific secondary antibodies, then washed and mounted (ProLong Gold Anti-Fade Reagent; Life Technologies). For immunodetection of FOXO1, a polymer-enhanced horseradish peroxidase–conjugated secondary antibody (EnVision+ system; Dako) was used and immunocomplexes were detected via tyramide–fluorescein isothiocyanate amplification (1:1000 for 3 min; Perkin-Elmer). For biotin-conjugated secondary antibodies (BCL6 or AID immunodetection), streptavidin-fluorochrome was added as a final step. Immmunofluorescence images were captured using a Nikon Eclipse E400 microscope and the NIS Elements software (Nikon). All images were colored, resized and merged using Adobe Photoshop software. Immunoblot analysis LZ and DZ human tonsillar GC fractions were sorted from mononuclear cell suspensions as previously detailed in (Victora et al., 2012). Cell suspensions were kept in ice at all times in PBS+0.5% BSA, and LZ and DZ fractions sorted into cold PBS-0.5% BSA. Purity of each fraction was confirmed to be >95%. Sorted cell fractions were lysed in 1% SDS lysis buffer (50 mM Tris, pH 8.0, 1mM EDTA, 100mM NaCl, 5mM DTT and 1%SDS) containing protease inhibitor cocktail, 1 mM PMSF, and phosphatase inhibitors: 50 mM sodium fluoride, 1 mM sodium orthovanadate, 10 mM beta-glycerophosphate; and were briefly sonicated using a Bioruptor device (Diagenode). Naïve B cells and total CD77+ GC B cells were isolated as previously described without modifications (Klein et al., 2003). The protocol for isolation of naïve B cells relies on capturing this population with anti-IgD antibodies, which could transiently induce modest activation of BCR signaling (Rickert, 2013). Protein lysates were resolved in 4–12% Tris-Glycine gels (Novex, Life Technologies) and transferred to nitrocellulose membranes, which were incubated with specific primary antibodies (Table S5) overnight at 4°C with gentle agitation. Horseradish peroxidase–conjugated secondary antibodies (Table S5) and ECL Reagent (Thermo) were used for detection. Luciferase reporter assays and DNA vectors The Ramos Burkitt Lymphoma cell line (ATCC CRL-1596) was maintained in Iscove’s Modified Dulbecco Medium (IMDM, Life Technologies) plus 10% FBS and 100μg/ml penicillin-streptomycin (5%CO2 and 37°C). The regions corresponding to the PRDM1-beta regulatory region (hg19_ chr6:106545152-106546111); CXCR4 5’ proximal enhancer (hg19_ chr2:136889207-136890467), and the CLOCK gene 5’ proximal promoter (hg19_chr4:56411577-56414042) were amplified from human normal genomic DNA (isolated from primary foreskin fibroblasts) using specific primer pairs and KAPA HiFi DNA polymerase (KAPA Biosystems). PCR products were subcloned into the pNL1.1 reporter vector (Promega) via Gibson Assembly (New England Biolabs). All constructs were further verified by Sanger Sequencing, and are freely available upon request. pcDNA3-BCL6 was constructed by subcloning a 2.4 Kb human BCL6 cDNA fragment into the BamHI/SmaI sites of pCDNA3.1. pCMV-FKHR (FOXO1) and pCMV-dnFOXO1(D256) were a kind gift of Drs. Megan Keniry and Ramon Parsons (Keniry et al., 2013). These plasmids were co-electroporated in Ramos B cells together with the above reporter constructs, using the Neon Transfection System (Life Technologies). Briefly, 10ul aliquots of a 7.6x106 cell/mL suspension were mixed with 500ng of total DNA and electroporated using a single 30ms pulse at 1350V (6-10 replicates per point). The electroporated cell pools were transferred to growth medium without antibiotics, and harvested 72 hours post-transfection. Nanoluc levels were determined using the Nano-Glo Luciferase Assay System (Promega), according to manufacturer’s instructions. P-values were calculated using T-test statistics (unpaired, two-tailed, unequal variance) from 6-10 independent technical replicates and 3 different experiments. Analysis of somatic hypermutation events in Vh186.2 mouse variable heavy-chain regions For the analysis shown in Figure 3, mice were immunized by a single intraperitoneal injection of

100μg NP-KLH precipitated in complete Freund's adjuvant (Sigma). 14 days after immunization, B cell enriched fractions were isolated as described above, and stained with specific antibodies to label GC B cell fractions. In preliminary experiments we noticed that, based on the levels of Foxo1 protein intracellular staining, the efficiency of Cg1-Cre-mediated recombination at the Foxo1 locus was incomplete and variable between animals (~22% of GC cells on average escaped Foxo1 deletion, range 10-35%). Therefore, we added an extra step of Foxo1 intracellular staining prior to cell sorting to isolate true Foxo1-null GC subpopulations. Genomic DNA from ~1-2 × 105 sorted GC B cells was extracted using the QIAamp DNA Micro Kit (Qiagen) as per manufacturer’s instructions, but introducing some minor modifications based on previous publications (Hansmann et al., 2010). Specifically, we increased the temperature of the proteinase K digestion step to 60oC, and extended it overnight. This was critical to reverse the formaldehyde crosslinks generated during cell fixation prior to intracellular staining of Foxo1, and to efficiently recover the genomic DNA. The isolated genomic DNA was used to first confirm the purity of all cell fractions (>90% of Foxo1 deletion in Foxo1-null GC B cell fractions by PCR). PCR amplification of variable heavy-chain region 186.2–joining heavy-chain region 2 segments (Vh186.2-JH2), which are predominant in GC responses to NP immunization (Jacob and Kelsoe, 1992), was done using 50 ng of genomic DNA with a previously described semi-nested PCR protocol (Schwickert et al., 2009) (primer sequences detailed in Table S6). Briefly, two sequential rounds of PCR were performed (first with outer primers FO, RO; then with inner primers FI, RI) for 25 cycles (94°C for 30 s, 55.5°C for 30 s, and 68°C for 90 s) using high-fidelity DNA polymerase (Accuprime Taq DNA polymerase, Life Technologies). Final PCR products were subcloned into pCR4-TOPO TA vector (TOPO TA cloning kit for sequencing, Life Technologies) and single clones (~48 per mouse) were sequenced using T7 and T3 primers. Variable heavy-chain gene sequences were analyzed using the HighV-Quest online tool (The International Immunogenetics Information System, IMGT) (Lefranc, 2011; Lefranc et al., 2009). Unique sequences (clones) matching the V186.2 gene (IGHV1-72*01) were identified and aligned to the mouse germline counterparts. Differences in the frequency of clones with W33L between groups were determined using the Mann-Whitney Test (GraphPad Prism v 6.0 software). JH4 intron sequences were amplified from genomic DNA of murine GC B cells sorted 14 days after NP-KLH immunization, as above. PCR amplification was performed using high-fidelity polymerase (KAPA HiFi, Kapa Biosystems) and gene specific primers (see Table S6 for sequences), for 35 cycles. Unique sequences were aligned to the consensus reference sequence in Cg1-Cre mice, which differs from the C57BL/6 germline sequence at 5 nucleotide positions (Sander & Rajewsky, personal communication) using the SHMTool (Maccarthy et al., 2009), and statistical significance determined using the Mann-Whitney test (Graphpad Prism, v 6.0). In vitro CSR assays To study the process of class-switch recombination in activated mouse B cells, we isolated enriched splenic B cell fractions from 12-14 week-old non-immunized mice (Foxo1+/+ and Foxo1L/L

littermates) using magnetic beads, as described above. Cells were plated at 2.5 x106 cells/mL in OptiMEM (Life Technologies) containing 1 uM HTN-Cre (TAT-Cre, Excellgen) or PBS as vehicle, and 200 uM Chloroquine (Sigma-Aldrich) (to increase the efficiency of intracellular delivery), and incubated for 45 minutes (5% CO2 and 37°C). TAT-Cre was then inactivated by addition of 10% serum. Cells were washed and resuspended in B cell medium (RPMI +15%FCS +55 uM beta-mercaptoethanol +10 mM HEPES +100ug/mL penicillin/streptomycin) containing 20 ug/mL lypopolysaccharide (LPS, Sigma-Aldrich) and 40 ng/mL mouse Interleukin-4 (IL-4, Preprotech). The culture medium was regularly replenished to avoid exhaustion. Three to four days (~85 hours) later, cells were harvested and processed for flow cytometry analysis (surface and intracellular staining) and RNA isolation. RNA extraction, cDNA synthesis and quantitative RT-PCR Total RNA from sorted GC B cell fractions (collected in PBS + 0.5%BSA + 2mMEDTA + 2% FBS) was isolated using the Nucleospin RNA XS RNA isolation kit (Macherey-Nagel), following the

manufacturer’s instructions. Linear Polyacrylamide (Ambion-Life Technologies) was used as a carrier. RNA integrity was assessed on a BioAnalyzer 2100 (Agilent), and samples with RNA integrity numbers >9 were processed for cDNA synthesis (10–25 ng of total RNA) using the Ovation RNA Amplification System (NuGEN) according to the manufacturer's instructions. This procedure yielded ~4–5 μg of cDNA (size range, 0.2–2 kilobases). A SYBR Green based PCR mix (Absolute Blue SYBR Green ROX mix; Thermo Scientific; or alternatively, SYBR Select Master Mix for CFX, ABI-Life Technologies) and gene-specific primers spanning at least one intron in the target gene were used for quantitative RT-PCR analysis (20–40 ng of cDNA per reaction). All reactions were performed in triplicate with a 7300 Real Time PCR System (Applied Biosystems). Results were calculated by the change-in-threshold (2−ΔΔCT) method, with Actb (encoding beta-Actin) as 'housekeeping' reference gene. Data are represented as fold change relative to the gene with lowest expression (arbitrarily set to 1), after normalization to Actb. Primer sequences are detailed in Table S6. Gene-expression profiling of murine GC B cells Total cDNA (3.75 μg), obtained as described above, labeled and fragmented with the Encore Biotin Module (NuGEN), was hybridized onto Mouse M430.2 microarrays (Affymetrix). Hybridization images were obtained with a GeneChip Scanner 3000 7G, connected to Command Console software (Affymetrix) at the Genomics Core in the Department of Pathology at Columbia University Medical Center. Raw analysis was done using the Affymetrix Expression Console Software. Gene-expression data were extracted and normalized with the MAS5.0 algorithm. Three samples per condition (Foxo1+/+, Foxo1+/L and Foxo1L/L, corresponding to 3 animals per genotype) were analyzed. Supervised gene-expression analyses were done using T-test statistics, selecting markers with a false discovery rate (FDR)≤0.05, and fold change≥1.5 fold. The results of these analyses are reported in Figure S1 and Table S1. Gene expression raw data has been deposited in the Gene Expression Omnibus (GEO) database (record GSE69216). Chromatin Immunoprecipitation (ChIP) GC B cells, purified from reactive tonsils as described (Klein et al., 2003), were cross-linked with 1% formaldehyde for 10 min at RT, quenched by the addition of glycine to a final concentration of 0.125 M, and frozen. ChIP was performed on two independent pools of GC cells, each comprising 3-5 donors. Cell lysis and nuclei isolation was performed using the TruChIP High Cell Chromatin Shearing Kit with SDS (Covaris). Nuclei were sonicated in the S220 Ultrasonicator (Covaris) in order to obtain chromatin fragments ranging 200 to 500bp. Sheared chromatin from about 5-10x106 cells was incubated over-night with 4ug of anti-BCL6 (N3, Santa Cruz), anti-FOXO1 (Abcam) or anti-H3K27Ac (Active Motif). Protein A magnetic beads were then added for 4hr incubation followed by sequential washes at increasing stringency and reverse cross-linking. Upon RNAse and proteinase K treatment, ChIP DNA was purified using the MiniElute Reaction Cleanup Kit (Qiagen) and quantified using the Quant-iT PicoGreen dsDNA Reagent (Life Technologies). ChIP-seq library preparation and sequencing ChIP-seq libraries were generated using the Illumina TruSeq ChIP Sample Prep Kit, starting from 4ug of ChIP or Input DNA as previously reported (Blecher-Gonen et al., 2013). Libraries were quantified using the KAPA SYBR FAST Universal qPCR Kit (KAPA Biosystems), normalized to 10nM, pooled and sequenced in an Illumina HiSeq 2000 instrument as single-end 100 bp reads, obtaining on average 25x106 reads/sample. ChIP-seq analysis Sequencing data were acquired through the default Illumina pipeline using CASAVA V1.8 software package. Reads were aligned to the human genome (UCSC hg19) for human GC B cells using the Bowtie2 Aligner v2.1.0 (Langmead et al., 2009) allowing for up to two mismatches. Duplicate reads mapping in exactly the same genomic locations were removed with SAMtools

v0.1.19 using the rmdup (remove duplicates) option (Li et al., 2009). Reads were normalized to total reads aligned (counts per million). Peak detection was done using ChIPseeqer v2.0 (Giannopoulou and Elemento, 2011) enforcing a minimum fold change of 2 between ChIP and input reads, a minimum peak width of 100 bp, and a minimum distance of 100 bp between peaks. The threshold for statistical significance of peaks was set at 10-5 for FOXO1, 10-12 for BCL6 and 10-15 for H3K27Ac. Peaks were considered overlapping if they shared at least 1bp. The ChIP-Seq raw data has been deposited in the Gene Expression Omnibus (GEO) database (record GSE68349). The results of these analyses are summarized in Tables S2 and S4. DNA binding motif enrichment Transcription factor DNA binding motif search was performed on a set of uniformly sized target sequences of 600bp centered on the genomic locations of peak mid-points. Enrichment was considered relative to an equally sized background set of 600bp sequences extracted randomly from the UCSC hg19 human genome. The background sequences had their GC content distribution controlled to match that of target sequences. The motifs are described by position weight matrices (PWM) extracted from the TRANSAC 2010 (Wingender et al., 1996) and JASPAR 2014 (Mathelier et al., 2014) databases. In addition we constructed BCL6 motifs from published, validated binding sites that were not present in the databases: B6BS/MO/M2/M00424 (Basso et al., 2010) and BCL6 (Niu et al., 2003; Pasqualucci et al., 2003; Phan and Dalla-Favera, 2004; Tunyaplin et al., 2004). Each motif PWM was used to generate a log-likelihood model that calculated whether any given motif could be found in a given search region using a sliding window to compare motif bases with the actual genomic sequence. Only motifs scoring at least 50% of their maximum score were considered. For each motif, we counted the number of target sequences that contained the motif and the number of random background sequences that contained the motif. We then used a cumulative hypergeometric distribution to calculate the probability of observing the given number (or more) of target sequences with the motif by chance, assuming there was no relationship between the target sequences and the motif. The parameter space was optimized to find sensitivity and specificity thresholds that best discriminated between the target sequences and the background. The results of this analysis are found in Table S3. Integrated gene expression and ChIP-Seq data analysis Previously published gene expression data of human DZ and LZ GC subpopulations (Victora et al., 2012) were normalized with MAS5.0 and probes were collapsed to genes by maximum standard deviation. The differential expression analysis, to be integrated with the ChIPseq data, was performed by Student’s T-test (p-value≤0.05). The results of the integrated analysis of gene expression and ChIPseq data are detailed in Tables S2 and S4. Gene Set Enrichment Analysis (GSEA) Gene Set Enrichment Analysis (GSEA) was performed using GSEA 2.0 (Subramanian et al., 2005) with 1000 gene permutations. The Molecular Signature Database (Subramanian et al., 2005) was used to perform pathway enrichment analysis using a hypergeometric distribution and limiting the output to the top 100 gene sets with a FDR q-value≤0.05. GSEA results for FOXO1-bound, BCL6-bound and FOXO1/BCL6 co-bound genes are detailed in Table S4.

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