ep400 deposits h3.3 into promoters and enhancers during ......transformation (fuchs et al., 2001)....

Article

EP400 Deposits H3.3 into
Promoters and Enhancersduring Gene Activation
Graphical Abstract

Highlights

d Double-variant H2AZ-H3.3-containing chromatin stimulates

transcription in vitro

d EP400 contributes to gene activation in vivo and in vitro

d EP400 is necessary for H2AZ and H3.3 deposition into

enhancers and promoters in vivo

d Purified EP400 exchanges both H2AZ andH3.3 into canonical

chromatin in vitro

Pradhan et al., 2016, Molecular Cell 61, 1–12January 7, 2016 ª2016 Elsevier Inc.http://dx.doi.org/10.1016/j.molcel.2015.10.039

Authors

Suman K. Pradhan, Trent Su, Linda

Yen, ..., Jacques Cote, Siavash K.

Kurdistani, Michael F. Carey

[email protected]

In Brief

Deposition of the histone variants H2AZ

andH3.3 accompanies gene transcription

in metazoans. Pradhan et al. show that

EP400 contributes to transcription on

variant chromatin in vitro and that EP400

knockdown reduces deposition of H2AZ

and H3.3 into promoters and enhancers

and decreases gene expression in vivo.

Accession Numbers

GSE73742

mailto:[email protected]

http://dx.doi.org/10.1016/j.molcel.2015.10.039

Please cite this article in press as: Pradhan et al., EP400 Deposits H3.3 into Promoters and Enhancers during Gene Activation, Molecular Cell (2016),http://dx.doi.org/10.1016/j.molcel.2015.10.039

Molecular Cell

Article

EP400 Deposits H3.3 into Promotersand Enhancers during Gene ActivationSumanK. Pradhan,1 Trent Su,1 Linda Yen,2 Karine Jacquet,3 ChengyangHuang,1 Jacques Cote,3 Siavash K. Kurdistani,1,2

and Michael F. Carey1,2,*1Department of Biological Chemistry, David Geffen School of Medicine, UCLA, 351A Biomedical Sciences Research Building, 615 Charles E.

Young Drive South, Los Angeles, CA 90095-1737, USA2The Molecular Biology Institute, UCLA, Paul D. Boyer Hall, 611 Charles E. Young Drive South, Los Angeles, CA 90095-1570, USA3Laval University Cancer Research Center, CHU de Quebec Research Center-Oncology, Hotel-Dieu de Quebec, 9 McMahon Street,

Quebec City, QC G1R 2J6, Canada

*Correspondence: [email protected]://dx.doi.org/10.1016/j.molcel.2015.10.039

SUMMARY

Gene activation in metazoans is accompanied by thepresence of histone variants H2AZ and H3.3 withinpromoters and enhancers. It is not known, however,what protein deposits H3.3 into chromatin or whethervariant chromatin plays a direct role in gene activa-tion. Here we show that chromatin containing acety-lated H2AZ and H3.3 stimulates transcription in vitro.Analysis of the Pol II pre-initiation complex on immo-bilized chromatin templates revealed that the E1Abinding protein p400 (EP400) was bound preferen-tially to and required for transcription stimulationby acetylated double-variant chromatin. EP400 alsostimulated H2AZ/H3.3 deposition into promotersand enhancers and influenced transcription in vivoat a step downstream of the Mediator complex.EP400 efficiently exchanged recombinant histonesH2A and H3.1 with H2AZ and H3.3, respectively, ina chromatin- and ATP-stimulated manner in vitro.Our data reveal that EP400 deposits H3.3 into chro-matin alongside H2AZ and contributes to gene regu-lation after PIC assembly.

INTRODUCTION

Chromatin poses a structural barrier to the RNA Polymerase II

(Pol II) transcriptional machinery. The process of gene activation

leads to the recruitment of chromatin modifying and remodeling

complexes that coordinate Pol II transcription with the remodel-

ing and eviction of nucleosomes (reviewed by Li et al., 2007). In

metazoans, the canonical histones at gene promoters and en-

hancers are replaced by specific histone variants, H2AZ and

H3.3, which correlate with active regulatory elements and tran-

scribed genes (Ahmad and Henikoff, 2002; Chen et al., 2013,

2014; Mito et al., 2005). Although the amino acid sequences of

histone variants differ from the canonical histones, their role in

activated transcription is unclear. Studies from the Felsenfeld

and Henikoff groups, and others, suggest a positive correlation

MOLCE

between occupancy of variant chromatin at the promoter and

transcriptional activity (Hardy et al., 2009; Jin et al., 2009; Millar

et al., 2006; Talbert and Henikoff, 2010). Moreover, activation of

transcription triggers H3.3 deposition (Schwartz and Ahmad,

2005). These correlations raised the important questions of

whether variant chromatin facilitates transcription or accom-

panies it and how variant chromatin is targeted to promoters

and enhancers in mammalian cells.

Our current understanding is that H2AZ and H3.3 are enriched

at the promoter and enhancer regions of active genes, although

research indicates that they also play roles in genomic stability

and DNA repair (Adam et al., 2013; Ray-Gallet et al., 2011;

Xu et al., 2012). H3.3 distributes throughout the coding regions

of genes (Goldberg et al., 2010). H3.3 is also present at the

promoters of developmentally regulated genes, which are typi-

cally silenced or weakly transcribed in embryonic stem cells

(Banaszynski et al., 2013). H3.3 is expressed throughout the

cell cycle, and it has been argued that Chd1 and Chd2 deposit

it into chromatin (Konev et al., 2007; Siggens et al., 2015). H3.3

deposition is facilitated by the histone chaperones Hira and

Atrx/Daxx (Banaszynski et al., 2013; Goldberg et al., 2010;

Szenker et al., 2011). H2AZ deposition, performed by SWR1 in

yeast and SRCAP and EP400 in human cells, plays a role in

gene regulation. (Gevry et al., 2007; Mizuguchi et al., 2004;

Ruhl et al., 2006), while the histone chaperone ANP32E facili-

tates H2AZ removal (Obri et al., 2014). Despite our growing

knowledge of variant chromatin across the genome, mechanistic

studies that define its function beyond correlations are lacking.

Additionally, given that H2AZ and H3.3 appear to be inserted

coordinately into nucleosomes during transcription, the question

of how they are linked to each other remains unclear as different

proteins apparently mediate their assembly.

The coordination of transcriptionwith histonemodification and

remodeling is critical to gene regulation because specific histone

modifications recruit distinct effector proteins that alter the chro-

matin landscape to facilitate different stages of Pol II function (Li

et al., 2007). To understand this issue, we have been using a

GAL4-VP16-responsive in vitro transcription system coupled

with immobilized template (IT) assays to capture and identify

pre-initiation complex (PIC) composition under various condi-

tions (Johnson et al., 2002). We identified a comprehensive list

of factors recruited in a GAL4-VP16-responsive model promoter

Molecular Cell 61, 1–12, January 7, 2016 ª2016 Elsevier Inc. 1

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mailto:[email protected]

http://dx.doi.org/10.1016/j.molcel.2015.10.039


system by using multidimensional protein identification technol-

ogy (MuDPIT) validated by immunoblotting (Chen et al., 2012;

Lin et al., 2011). These included transcription factor IID (TFIID)

and the general transcription factors (GTFs) (transcription factor

IIB [TFIIB] and transcription factors IIE, IIF, and IIH), Mediator

complex, Pol II and its elongation complexes (the Cdk9-contain-

ing P-TEFb and PAF), together with the chromatin modification

factors p300, SAGA, Tip60 and Set1 complex, and the ATP-

dependent chromatin remodeling proteins Chd1, Ino80, and

EP400. Among these, the factors segregate into two categories:

those whose recruitment depends on Mediator, including GTFs

and TFIID, and those that are activator dependent but Mediator

independent, including SAGA and Tip60-EP400 (Chen et al.,

2012). Indeed, SAGA does not participate in PIC assembly but

acts afterward to allow chromatin transcription in vitro.

EP400 is a SWR1-class ATP-dependent chromatin remodel-

ing protein originally identified as an interacting partner for the

adenovirus E1A protein required for viral replication and cellular

transformation (Fuchs et al., 2001). EP400 is a subunit of the

Tip60-EP400 complex comprising �16 subunits, including the

Tip60 histone acetyltransferase (HAT), the phosphatidylinositol

3-kinase family protein kinase homolog TRRAP, and Brd8 (Cai

et al., 2003; Doyon and Cote, 2004). The EP400 H2AZ exchange

potential has been reported to play a role in regulated gene

expression (reviewed by Venkatesh and Workman, 2015) and

DNA repair (Xu et al., 2012). Tip60-EP400 also regulates embry-

onic stem cell identity (Fazzio et al., 2008) and cell cycle (Chan

et al., 2005; Tyteca et al., 2006). EP400 is associated with

gene promoters (Fazzio et al., 2008). Although EP400 is primarily

considered a subunit of the Tip60-EP400 complex, it also resides

within another complex that lacks Tip60 and is involved in

repression (Fuchs et al., 2001; Park et al., 2010).

To understand the role of histone variants in transcription, we

recreated GAL4-VP16-stimulated transcription by acetylated

chromatin bearing H2AZ and H3.3 in vitro. We captured PICs

on variant chromatin and demonstrated that they were enriched

in EP400. We validated our biochemical data in a doxycycline

(Dox)-inducible Tet-VP16 dependent reporter system and

genome-wide in U2OS cells. Knockdown (KD) of EP400 led to

a reduction in transcription and deposition of both H2AZ and

H3.3. Histone exchange experiments revealed that EP400 could

exchange canonical histones with both H2AZ and H3.3 in vitro.

Our data suggest that EP400 mechanistically links variant his-

tone deposition to gene transcription.

RESULTS

Variant Chromatin Stimulates Transcription In VitroTo understand how variant chromatin influenced transcription,

we generated chromatinized promoters in vitro bearing H2AZ-

H2B dimers, H3.3-H4 tetramers, or both and identified condi-

tions under which they stimulated transcription. We refer to the

chromatin as double variant or single variant depending on

whether it contains both H3.3 and H2AZ or each alone. We

took into consideration that transcriptionally active chromatin

is heavily acetylated in vivo. In proteomic analyses, H3.3 is en-

riched in H3K9 and H3K18 acetylation relative to H3.1 in Kc cells

(McKittrick et al., 2004). To provide sufficient levels of histone

MOLCEL 563

2 Molecular Cell 61, 1–12, January 7, 2016 ª2016 Elsevier Inc.

acetylation, we pre-acetylated the chromatin with recombinant

Gcn5 and Esa1, the catalytic subunits of yeast SAGA (acetylates

H3 and H2B) and NuA4 (acetylates H4 and H2A), respectively,

the latter being the homolog of human Tip60 (Doyon and Cote,

2004; Grant et al., 1997).

In the absence of HATs, GAL4-VP16 stimulated transcription

to similar levels on canonical and variant chromatin (Figure 1A,

lanes 2, 5, 8, and 11). However, after histone acetylation, the

double-variant chromatin displayed a highly reproducible stimu-

lation of transcription versus canonical (Figure 1A, lane 12 versus

lane 3). Because canonical chromatin has been widely used in

and is highly active for in vitro transcription, we were expecting

a transcription stimulation rather than absolute dependence on

variant chromatin. Importantly, the stimulation was significantly

weaker on single-variant chromatin bearing either H2AZ (lane

6) or H3.3 (lane 9) with the combination (lane 12) eliciting the

greatest effect. These data show that double-variant chromatin

directly stimulates transcription upon histone acetylation. The

ability to recreate transcription stimulation by double-variant

chromatin provided an assay to identify factors that mediate

the effect.

EP400 Is a Variant Chromatin-Specific TranscriptionFactor In VitroTo identify a specific factor that was either recruited or enriched

on acetylated variant chromatin, we used the IT assay to capture

an activator-responsive PIC from HeLa nuclear extract (Johnson

et al., 2002). We used immunoblotting of representative subunits

of all the PIC components identified by our previous MuDPIT

study to determine if any of them were enriched on double-

variant chromatin (Figure 1B) (Chen et al., 2012; Lin et al.,

2011). Without chromatin acetylation, GAL4-VP16 stimulated

recruitment of the factors identified by MuDPIT including GTFs,

TFIID, Mediator complex, Pol II, and others at nearly identical

levels on templates bearing canonical or double-variant chro-

matin (Figure 1B). In contrast, EP400, a component of the

Tip60 HAT complex, was enriched by an average of 2.5-fold

(Figure 1B) on acetylated double-variant versus acetylated

canonical chromatin (graphed in Figure 1C). Although Brd8,

another Tip60 complex subunit, was enriched on acetylated

double-variant chromatin, oddly the Tip60 HAT subunit was

not (Figure 1B). We conclude that EP400 enrichment within a

PIC is stimulated by acetylated double-variant chromatin.

Histone acetylation did not typically alter the amounts of

histones bound to the immobilized chromatin templates. The

amounts of chromatin-bound H3.3 and H3.1, along with the

amounts of H2B, used as a measure of dimers with H2AZ or

H2A, were not greatly affected by acetylation (Figure 1B, bottom).

Furthermore, acetylation did not cause H3.3 or H3.1 bound (B) to

the IT todissociate into the supernatant andwash (S) asmeasured

by a pan-acetyl H3 antibody (Figure 1D). Additionally, H3.3 and

H3.1 were acetylated at similar levels. We conclude that neither

the transcriptional stimulatory effect of the acetylated double-

variant chromatin nor the enhancedbinding of EP400 is due to dif-

ferences in acetylation or instability of acetylated chromatin.

To investigate EP400’s role in transcription, we immunode-

pleted it (EP400D) from HeLa extracts (Figure 1E) and examined

if its loss affected PIC assembly or transcription of acetylated

9

A B

C

D

E

F

G H

Figure 1. Variant Chromatin Stimulates Transcription In Vitro in an EP400-Dependent Manner(A) In vitro transcription. End-biotinylated G5E4T was assembled into chromatin using canonical, single-variant, or double-variant octamers as indicated and

immobilized on paramagnetic beads. Chromatin was acetylated by Gcn5 and Esa1 (HAT) as indicated and incubated with nucleotides (NTPs) in HeLa nuclear

extract with or without GAL4-VP16 (Activator). A phosphorimage of the primer-extension gel is shown and graphed using mean and SD spectral units of 3

replicates. **p < 0.01 by Student’s t test. n.s., not significant.

(B) IT capture of PICs. Immobilized canonical or double-variant chromatin was as in (A) minus NTPs. Purified PICs were immunoblotted with antibodies to

indicated proteins.

(C) Quantitation of select proteins from immunoblot in (B) of acetylated canonical or double-variant chromatin.

(D) Levels of H3 acetylation measured for canonical and double-variant chromatin using pan-H3Ac antibody: B (bound) and S (supernatant/wash) fractions.

(E) EP400 immunoblots of Mock (M) and EP400-depleted (EP400D) extracts over a 9-fold titration range.

(F) IT analysis of select PIC components in Mock and EP400D extracts.

(G) Silver-stained gel of EP400 preparation.

(H) In vitro transcription of acetylated chromatin in Mock or EP400D extracts. Graphed as in (A) for 3 replicates but with or without recombinant EP400 as

indicated. **p value < 0.01 by Student’s t test.


double-variant versus canonical chromatin. Although EP400D

did not affect the recruitment of representative transcription

factors to the PIC (Figure 1F), we observed a reproducible and

significant decrease in transcription from variant chromatin.

Addition of recombinant EP400 (Figure 1G) restored the tran-

scriptional stimulation from double-variant chromatin in the

depleted extracts with little effect on canonical chromatin (Fig-

MOLCE

ure 1H). The data in Figures 1F and 1H suggest that EP400

acts independent of PIC assembly to facilitate transcription on

acetylated double-variant chromatin.

EP400 Affects Transcription In VivoThe in vitro data prompted us to investigate whether EP400

contributes to transcriptional activation in a cell-based system.

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A B D

C

E F

Figure 2. EP400 Is Necessary for Transcription in a Model Cell-Based Reporter System

(A) Schematic of U2OS Tet-On VP16 reporter system.

(B) Quantitation of F-Luc RT-PCR products at indicated time points (hours after Dox induction [h.p.i.]) in untreated, Mock, and EP400 siRNA-KD cells 72 hr after

transfection of siRNA.

(C) ChIP-quantitative PCR (qPCR) of promoter at the indicated time points with antibodies against VP16, Pol II, MED1 (Mediator), EP400, and H3.3. Bar graph

indicates enrichment at time points relative to input DNA.

(D) EP400 immunoblot of Mock versus siRNA KD normalized via GAPDH levels.

(E) Immunoblot analysis of H2AZ, H3.3, H3, and H4 for crosslinked chromatin fractions or whole-cell extracts. The numbers indicate the intensity of signal in KD

normalized to 1 for Mock (M); representative blots are shown.

(F) ChIP of Pol II, Mediator, and H3.3 in Mock versus EP400 KD cells time post Dox. **p < 0.01 by Student’s t test.


We used a U2OS cell line bearing >10 integrated copies of the

Dox-inducible Tet-VP16-responsive luciferase reporter gene

(Black et al., 2006; Lin et al., 2011) (Figure 2A). Time course

experiments showed that reporter gene mRNA accumulated

in a gradual manner within an 8 hr window (Figure 2B). Locus

specific chromatin immunoprecipitation (ChIP) experiments

at the promoter of the reporter gene (Figure 2C) detected

reproducible enrichment of Tet-VP16, Pol II, Mediator, and

EP400. We conclude that EP400 is recruited to the promoter

in a VP16-responsive manner in vivo similar to its recruitment

in vitro.

MOLCEL 563


We next examined whether EP400 is important for reporter

gene activation in vivo. EP400was knocked down by small inter-

fering RNA (siRNA) for 72 hr, and gene activation was compared

with Mock siRNA-treated cells. Immunoblot analysis revealed a

�4-fold EP400 KD at low (13) and high (33) siRNA amounts,

as reported (Mattera et al., 2010). Under KD conditions, we

observed a 6-fold decrease in reporter gene expression as

compared to Mock-treated cells (Figure 2B). Because EP400

KD is known to affect H2AZ incorporation in vivo (Gevry et al.,

2007, 2009; Lee et al., 2012), we analyzed chromatin-associated

levels of H2AZ alongside H3, H4, and H3.3. H2AZ levels

9

A

B C

D

E

Figure 3. Genome-Wide Analysis of EP400

and Variant Chromatin at Promoters

(A) Heatmaps of EP400, Pol II, Mediator (MED26),

H3.3, and H2AZ at U2OS promoters (3 kb flanking

TSSs). P values for enrichment are plotted and

ranked by EP400. ThemRNA heatmaps plot FPKM

of Mock alongside fold change (FC) of log2 KD/

Mock FPKM (green is downregulated, red is up-

regulated, and black is no change). P value of dif-

ferences by Wilcoxon rank-sum test = 9 3 10�19.

(B) Representative genome browser view.

(C) Metagene analyses of (A) clustered by gene

expression. The colored lines indicate enrichment

for each protein of top (red; C1), middle (green; C2),

and bottom (purple; C3) 10% of expressed genes;

black line (C4) is average for all genes.

(D) Fold change in enrichment upon EP400 KD for

ChIP experiments in (A) plotted as log2 (KD/Mock)

on EP400-bound gene promoters by significant

peaks (TSS ± 3 kb). Green indicates reduced

binding of indicated protein. Total H3 (H3) ChIP

data are added to this plot but not shown in (A).

(E) Boxplots of �log2 Poisson p values of H3.3,

total H3, H2AZ, Pol II, and MED26 ChIP signals in

M and KD conditions. Error by Wilcoxon rank-sum

test is shown.


decreased in chromatin from EP400 siRNA-treated cells, but

strikingly, we observed a greater decrease in H3.3 (Figure 2E).

By contrast, the levels of H2AZ and H3.3 in whole-cell extract

were relatively unaffected (Figure 2E).

The decrease of H3.3 was unexpected. We therefore

measured H3.3 accumulation at the reporter gene promoter

and found that it was deposited as a time-dependent function

of gene activation (Figure 2C). However, under EP400 KD condi-

tions, histone variant H3.3 deposition was significantly compro-

mised along with Pol II recruitment, whereas the binding of

Tet-VP16 (data not shown) and Mediator remained unchanged

(Figure 2F). These data suggest that EP400 is necessary for

transcription on variant chromatin and may contribute to H3.3

deposition. This observation is important because little is known

about EP400’s role in gene regulation. To determine whether our

observation was a nuance of our reporter gene or a global phe-

nomenon, we next asked whether the model template results

were paralleled genome-wide.

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Molecular Cell 61, 1

EP400 Is Found at the Promoter andCoding Regions Genome-wide andCorrelates with H3.3 and GeneExpressionTo perform the genome-wide analysis,

we carried out gene expression mea-

surements (RNA sequencing) and ChIP

sequencing (ChIP-seq) in the same

U2OS cell line as the model reporter

gene. Pol II, Mediator, EP400, and the

histone variants H3.3 and H2AZ were

compared at the promoter under Mock

and EP400 KD conditions. To gauge the

effectiveness of the parallel ChIP experi-

ments, we generated heatmaps sorted

from high to low EP400 binding by p values of the enrichment.

The heatmaps show that under Mock conditions, the rank

enrichment of EP400 correlates roughly with that of Pol II, Medi-

ator, H3.3, and H2AZ (Figure 3A). Importantly, the enrichment of

promoter-bound Mediator, Pol II, EP400, H2AZ, and H3.3 ap-

pears somewhat proportional to the rank abundance of mRNA.

Pol II and Mediator are concentrated near the promoter. H3.3

and H2AZ peaked in a bifurcated manner around the promoter,

but H3.3 extended outward into the gene body, as did EP400

(Figures 3A and 3B). The extension of H3.3 into gene bodies

has been noted previously (Goldberg et al., 2010; Jin et al.,

2009). Indeed, metagene analysis with genes binned by mRNA

levels corresponding to the top (Figure 3C1), middle (Figure 3C2),

and bottom (Figure 3C3) 10% of genes demonstrated that H3.3

and EP400 enrichment in the promoter and gene body rank-

correlated with gene expression (Figure 3C).

Remarkably, EP400 KD elicited a significant and reproducible

negative effect on H3.3 enrichment (Figure 3A, compare Mock

–12, January 7, 2016 ª2016 Elsevier Inc. 5

2E-120

DOWN4024

UP2086

3854

Genes Downregulated

CAGenes

Upregulated

D

Mock KDLo

g 2(F

PK

M)

0 5 10 15 20 25

Mitosis

Chromatin organization

Cell cycle

RNA processing

Translation

-log10(p value)

15

10

5

0Mock KD

15

10

5

0

B

M KD

2E-120

0.0

5.0

0.0

5.0

M KD

Genes Downregulated

Genes Upregulated

3E-155

Log2(FPKM)

2E-120

Log2(FPKM)

Figure 4. Analysis of EP400 KD on Variant Chromatin Gene Expression

(A) Venn diagram of differentially expressed genes in EP400KD versusMock. RNAs from the top 9,964 genes (by >1 FPKM value) analyzed for changesR1.5 fold;

4,024 were downregulated (green) and 2,086 (red) were upregulated in EP400 KD conditions.

(B) Boxplot representing downregulated (green) and upregulated (red) genes by FPKM in M and KD conditions using a log2 scale.

(C) Heatmaps showing the downregulated (green) and upregulated (red) genes in M and KD by FPKM and ranked by fold change for clarity.

(D) Gene Ontology analysis of affected genes by category. P values were corrected for multiple hypothesis testing using Benjamini correction.


with KD). The data are further supported by heatmaps of fold

change in Figure 3D, ranked on a promoter-by-promoter basis

by EP400 abundance (significant peaks). The columns show a

decrease in abundance of the indicated proteins upon EP400

KD. EP400 KD elicited the most evident change on H3.3

enrichment. The KD also decreased Pol II and H2AZ enrichment

with less change in MED26. Total H3 levels at promoters, as

measured by pan-H3 antibody ChIP, decreased far less than

H3.3. These data suggest but do not prove the possibility that

the loss of H3.3 is being compensated for by increased retention

of H3.1. Note from the boxplots of Figure 3E that the decreased

enrichment of all proteins under KD conditions is significant by

Wilcoxon rank-sum tests. However, the p values ranking from

lowest to highest are H3.3, Pol II, H2AZ, and total H3, with the

highest p value being MED26. The KD elicited effects on gene

expression for many genes, as illustrated in the heatmap

showing the log2 ratio of KD/Mock gene expression (Figure 3A;

green is downregulated and red is upregulated); we discuss

these data further below. In conclusion, our genome-wide anal-

ysis in the presence of EP400 siRNA is largely consistent with our

model reporter results showing that decreased EP400 levels

lead to a decrease in H3.3 deposition but have a much smaller

effect on Mediator.

As predicted from themodel reporter data, EP400 KD affected

gene expression (Figure 4A). Notably, among the top �10,000

genes ranked by fragments per kilobase per million mapped

reads (FPKM) greater than 1, 6,110 genes are affected R1.5-

fold upon KD; 4,024 genes are downregulated, and 2,086 are

upregulated (Figure 4A). The boxplot analysis in Figure 4B dem-

onstrates that in Mock cells, the genes downregulated upon KD

express �10-fold more mRNA (by median) than the upregulated

MOLCEL 563


ones, suggesting that EP400’s predominant role in the cell is as a

positive transcriptional regulator. The upregulated genes might

be due to repression by EP400, which is observed in some con-

texts (Fazzio et al., 2008; Tyteca et al., 2006). Also, H3.3 is found

onweakly transcribed genes bound by PRC2 (Banaszynski et al.,

2013). The heatmaps in Figure 4C directly show the effect of KD

on downregulated (green) and upregulated (red) genes by

comparing side by side the FPKM in Mock and KD.

Gene Ontology analysis identified translation, RNA process-

ing, cell cycle, chromatin organization, and mitosis as the top

hits for the functional enrichment of EP400-bound genes (p <

10�10). Such a result might be expected for a highly proliferative

cancer cell line and is consistent with EP400’s binding to the viral

oncoprotein E1A during cellular transformation (Fuchs et al.,

2001) and the EP400-mediated cell cycle effects (Chan et al.,

2005; Tyteca et al., 2006) (Figure 4D). We conclude the EP400

is required for full expression of a subset of highly transcribed

genes but is also necessary for repression of fewer genes, with

the caveat that some effects may be indirect (i.e., cases in which

EP400 might positively or negatively regulate a specific tran-

scriptional repressor).

EP400 Is Enriched at Enhancers and Regulates H3.3OccupancyTip60-EP400participates inH2AZexchange at enhancers (Gevry

et al., 2009). We therefore analyzed EP400 distribution at en-

hancers identified by the presence of both the H3K4me1 and

H3K18ac histone modifications under Mock conditions. H3K18

is acetylated by the p300 and CBP HATs (Jin et al., 2011). We

restricted our analysis to distal intergenic enhancers located

R3 kb upstream or downstream of an annotated transcription

9

Figure 5. Analysis of EP400 KD on Enhancer Occupancy and Gene Expression

(A) Heatmaps of H3K4me1, H3K18ac, EP400, H3.3, H2AZ, and Pol II at active distal intergenic U2OS enhancers (±5 kb from the center of H3K4me1 peaks) in KD

and/or Mock cells. Active enhancers were scored by the H3K4me1 and H3K18ac marks, excluding +3 kb upstream of TSS or downstream of TTS, and sorted by

H3K4me1 enrichment in Mock and EP400 siRNA KD cells.

(B) Browser track of putative enhancers. Those showing markers consistent with active enhancers are indicated by arrows below the tracks (i.e., identifiable

peaks of Pol II, Mediator, EP400, H3.3, H2AZ, H3K18ac, and H3K4me1).

(C) Fold change in enrichment of H3.3, H2AZ, and Pol II as in Figure 3D for all EP400-bound enhancers by significant peaks, ranked by H3K4me1 abundance.

Wilcoxon rank-sum p values for change in H3.3 and H2AZ are both <2 3 10�308 (saturated), and p value for Pol II is 2 3 10�148.

(D) Boxplots of �log2 Poisson p values of H3.3, H2AZ, and Pol II in M and KD conditions for top 10% enhancers with p values.

(E) Nearest neighbor effects by GREAT analysis. Average FPKM was plotted for nearest enhancer-proximal genes for top, middle, and bottom 10% of putative

enhancers as sorted by H3K4me1 enrichment.


start site (TSS) or transcription termination site (TTS). When

the enrichment p values are sorted by the relative abundance

of H3K4me1, EP400 correlates with the presence of H3K18ac,

H3.3, and Pol II (Figure 5A), consistent with the browser track

(Figure 5B).

The fold change (Figure 5C) and boxplot (Figure 5D) analyses

of the relationships reveal a strong decrease in H3.3 levels

upon EP400 KD. The effect of EP400 KD on H2AZ levels was

also evident by the enrichment heatmaps (Figure 5A), fold-

change heatmaps (Figure 5C), and boxplots (Figure 5D), consis-

tent with a previous report (Gevry et al., 2009). Pol II occupancy

also decreased at enhancers upon EP400 KD, although its over-

all enrichment at enhancers was lower than promoters (note

scales used Figure 5A versus 3A).

The Genomic Regions Enrichment of Annotation Tool (GREAT)

was used to identify the nearest neighboring genes to enhancers.

The mRNA FPKM values from the enhancer-neighboring genes

decrease upon EP400 KD cells (Figure 5E). Collectively, our

MOLCE

data establish a link between EP400 and H3.3 occupancy

at both promoters and enhancers and suggest that EP400 bound

at enhancers might contribute to full levels of expression

from the nearest neighboring genes. However, without estab-

lishing a direct enhancer-gene relationship, the data are simply

correlative.

H2AZ and H3.3 Exchange Activity of EP400 In VitroThe data from the genome-wide study raised the possibility

that EP400 is necessary for H3.3 deposition during transcrip-

tion. It is difficult to address in vivo whether H3.3 deposition is

a direct effect of EP400 or an indirect effect due to other

EP400-affected transcription steps that, in turn, influence

histone deposition. However, others have shown that EP400

mediates H2AZ exchange in standard in vitro assays (Gevry

et al., 2007). In order for an H2AZ-H3.3 double-variant nucle-

osome to be deposited during gene activation, it would have

to replace an entire canonical nucleosome. We imagined two

L 5639


A

B

H2A

Z on

Bea

dsH

3.3

on B

eads

Input(30%)

H3.3 TetramerATP

EP400

Input(30%)

H2AZ on beads

H2AZ in sup

H2B on beads

H2AZ DimerATP

EP400

Flag H3.3H3.1Flag H3.3H3.1

54321

12

8

4

0H

3.3

on B

eads

ATPEP400

BeadsFlag-H3.3

C

H3.3 TetramerATP

EP400

Flag H3.3H3.1

H3.1Flag H3.3

Beads

Sup

H2A on beads

H2A in sup

H2A DimerATP

EP400

H2AZ on beads

H2AZ in sup

H2AZ DimerATP

EP400

Input(30%)

Input(30%)

Input(30%)

D.I H2AZ vs H2A Exchange

E.I H3.3 vs. H3.1 Exchange

D.II H2A vs H2AZ Exchange

H3.3/H2AZ Octamer

DNA Chromatin

Beads

Sup

H3.1 TetramerATP

EP400

Flag H3.3H3.1

H3.1Flag H3.3Input

(30%)

E.II H3.1 vs. H3.3 Exchange

H3.3 TetramerATP

EP400

His-H3.3Flag-H3.3

Flag-H3.3His-H3.3Input

(30%)

E.III H3.3 vs. H3.3 Exchange

Beads

Sup

Beads

Sup

4

321

0

H2A in sup

0

Figure 6. Histone Exchange by EP400 In Vitro

(A and B) Three hundred nanograms immobilized canonical chromatin was incubated as indicated with Apyrase-treated EP400 and 300 ng H3.3-H4 tetramers or

H2AZ-H2B dimers ± 1mMATP. Chromatin was captured, washed, and subjected to immunoblotting for H2AZ (A) or H3 (B). FLAG-H3.3 is distinguished fromH3.1

by mobility and detected using a pan H3 antibody; H2B on beads as a control.

(C) Alternatively, 150 ng of immobilized naked DNA or 300 ng canonical chromatin was incubated with EP400 and 300 ng of histone variant octamers as above

and FLAG-immunoblotted for H3.3. Graphs in (A–C) represent R3 independent experiments. Input and conditions for (D) and (E) were as above.

(D) H2AZ exchange into bead-bound H2A-containing chromatin (I); reverse reaction of H2A deposition into H2AZ chromatin (II).

(E) H3.3 exchange into H3.1 chromatin (I); reverse reaction of H3.1 deposition into H3.3 chromatin (II); exchange of His-H3.3 into Flag-H3.3 chromatin (III).


ways that a nucleosome could be replaced. In one model,

the presence of a canonical nucleosome would stimulate the

swapping out with a variant nucleosome. In the other, the

canonical nucleosome would be evicted by an alternative pro-

cess, which would then create space for the assembly of the

histone variants on naked DNA. The relevant in vitro predic-

tions of the two models are that histone variants would be

MOLCEL 563


deposited onto a template in either a chromatin or DNA stim-

ulated manner.

To test these predictions directly, we incubated magnetic

bead-ITs, bearing either canonical chromatin or naked DNA,

with free variant H2AZ-H2B dimers, H3.3-H4 tetramers, or dou-

ble-variant octamers in the presence of recombinant EP400 plus

or minus the addition of ATP (Figure 6). As previously noted

9


(Gevry et al., 2007), EP400 co-purifies with ATP, and the enzyme

Apyrase was necessary to reduce exchange activity in the

absence of added ATP. Although a modest amount of back-

ground binding by the histone variants was to be expected,

EP400 stimulated the reproducible deposition of both H2AZ-

containing histone dimers (Figure 6A) and H3.3 tetramers

(Figure 6B) onto canonical nucleosome-bound beads in an

ATP-stimulated manner. ATPgS did not support the exchange.

Importantly, we detected clear H2A and H3.1 eviction into the

supernatant, in parallel to the deposition of H2AZ and H3.3

(Figures 6A and 6B). In contrast, EP400 deposited variant chro-

matin less efficiently on a naked DNA template compared with a

canonical chromatin template (Figure 6C). This result is in agree-

ment with the idea that EP400 functions via interaction with chro-

matin (Ruhf et al., 2001). Although EP400 could perform the

reverse exchange of H2A and H3.1 into variant chromatin, its

activity was substantially lower in side-by-side reactions than

exchange of histone variants into canonical chromatin (compare

Figure 6D.I for H2AZ with Figure 6D.II for H2A; compare Fig-

ure 6E.I for H3.3 with Figure 6E.II for H3.1). In contrast, EP400

efficiently exchanged a His-tagged H3.3 onto Flag-tagged

H3.3-containing chromatin (Figure 6E.III). We conclude that

assembly of variant chromatin is stimulated by the presence of

chromatin on the template and proceeds in a direction that fa-

vors deposition of histone variants in place of canonical ones.

Our data confirm previous reports that EP400 is an H2AZ ex-

change factor and, importantly, extend that functionality to an

H3.3 exchange factor and provide a plausible explanation for

how H3.3 is deposited during transcription.

DISCUSSION

Our study shows that (1) acetylated double-variant chromatin is

stimulatory for transcription in vitro in an EP400-dependent

manner (Figure 1), and (2) an EP400 KD-dependent decrease

in deposition of H3.3, and to a lesser extent H2AZ, leads to de-

fects in enhancers and promoters that primarily result in lower

levels of transcription in vivo (Figures 3, 4, and 5). The KD elicited

a widespread decrease in genomic mRNA ranging from 1.5- to

20-fold for �4,000 genes (median effect �2.4-fold) (Figure 4).

The decrease in expression of the integrated Tet-VP16 respon-

sive reporter gene fell within this range (Figure 2). The results

from our in vitro transcription system using both Mock and

EP400-depleted extracts are particularly relevant because they

represent direct effects and as such implicate EP400 and dou-

ble-variant chromatin in stimulation of transcription (Figure 1).

We did note many upregulated genes upon KD, suggesting

that these may be repressed, but their mRNA expression levels

were lower, and they represented half the number of genes posi-

tively regulated by EP400.

Acetylation of double-variant chromatin by Gcn5 and Esa1 is

required for EP400-dependent transcription stimulation in

HeLa extract. This is relevant because Gcn5, as part of SAGA,

and Tip60, the human homolog of Esa1 and the HAT subunit of

the Tip60-EP400 complex, are the major HATs stably recruited

to PICs in vitro by our model activator GAL4-VP16 (Chen et al.,

2012). The SAGA and Tip60-EP400 HAT complexes are re-

cruited by activator in a Mediator-independent manner and are

MOLCE

not required for assembly of Pol II and the GTFs at a promoter.

SAGA functions downstream of PIC assembly by enabling effi-

cient transcription (Chen et al., 2012). EP400, as a subunit of

the Tip60-EP400 complex, functions analogously to SAGA in

that its loss does not greatly affect Mediator, GTF, or Pol II

recruitment at the promoter in vitro but does affect transcription

specifically on acetylated double-variant chromatin (Figure 1).

These data suggest that like SAGA, EP400 functions down-

stream of PIC assembly by facilitating initiation or elongation

by Pol II. Indeed, on both our model reporter template and

genome-wide, Mediator recruitment, which is central to PIC as-

sembly, was far less affected by EP400 KD than was H3.3.

The yeast homologs of SAGA and Tip60 (NuA4) are necessary

for the efficient recruitment of the SWI family member RSC to

genebodies inS. cerevisiae (Spain et al., 2014). In turn,RSCplays

a role in both recruitment of Pol II to genebodies and transcription

elongation (Carey et al., 2006; Spain et al., 2014). Indeed, RSC

binds with high avidity to chromatin acetylated by Gcn5 and

Esa1 probably through one or more of its bromodomains. By

analogy, the SWI-like EP400 displays a 2.5-fold enrichment on

acetylated double-variant chromatin and the Tip60-EP400 com-

plex contains a bromodomain factor Brd8, which is enriched

similarly (Doyonet al., 2004). It is plausible thatBrd8bindsdirectly

to acetylated histones and is responsible for targeting EP400 to

acetylated double-variant chromatin in vivo.

Our in vitro data show that EP400 has the ability to exchange

or deposit variant histones onto chromatin more efficiently

than naked DNA, suggesting that its role is to deposit H3.3 into

canonical chromatin during gene transcription (Figure 6). In

side-by-side comparisons in vitro, EP400 exchanged variant

histones into canonical chromatin more effectively than canoni-

cal histones into variant chromatin. However, interestingly, a His-

tagged H3.3-H4 bearing tetramer was exchanged very efficiently

into double-variant chromatin bearing Flag-tagged H3.3. Thus,

the reaction seems to favor insertion of soluble H3.3 irrespective

of whether the chromatin is canonical or variant. This observa-

tion might suggest that EP400 exchanges H3.3 present in the

crude HeLa nuclear extracts for the chromatin bound H3.3.

Perhaps this continuous exchange process favors a chromatin

state that stimulates transcription in vitro.

The discovery of EP400 at both promoters and enhancers

correlated with the presence of double-variant nucleosomes

(Gevry et al., 2009; Taubert et al., 2004). Because EP400 is a sub-

unit of the Tip60 complex, it is likely recruited to the DNA in part

through activators interacting with the TRRAP subunit of Tip60,

which is also present in SAGA (Vassilev et al., 1998), and in

part through its interaction with chromatin, possibly through

Brd8. Thus, the TRRAP subunit likely contributes to recruitment

of both the Tip60 and SAGA HATs to enhancers and promoters

allowing effective activator-targeted acetylation of all four his-

tones (Brown et al., 2001). Because numerous activators recruit

Mediator and SAGA individually through distinct subunit interac-

tions, it makes sense that SAGA and Tip60-EP400 could function

independently of Mediator but be necessary for its ability to

generate productive mRNA synthesis in a chromatin environ-

ment. Indeed, the separate recruitment events may ensure the

fidelity of combinatorial control as a mechanism for differential

gene regulation on chromatin (Carey, 1998).

L 5639



A similarity shared among promoters and enhancers is the

strong effect EP400 KD has on H3.3 deposition. The KD has a

smaller effect on deposition of H2AZ. Interestingly, the effect of

the KD on H2AZ enrichment was apparent at enhancers bearing

high levels of H3K4me1 and was paralleled by the effect on Pol II

(Figure 5). We presently do not understand this phenomenon but

note that the H2AZ enrichment is lower at enhancers containing

high levels of H3K4me1 and Pol II enrichment is lower at en-

hancers compared with promoters. Although we observed a

mild decrease in H2AZ upon EP400 KD, we note that the meta-

zoan homolog of yeast SWR1 SRCAP also exchanges H2AZ for

H2A in vitro and in vivo. It is plausible that SRCAP is redundant

with EP400 accounting for the smaller effect of KD on H2AZ

versus H3.3 (Wong et al., 2007).

Chd1 and Chd2 have been reported to control H3.3 incorpora-

tion at active chromatin in metazoans (Konev et al., 2007;

Siggens et al., 2015). Chd1 bears a SWI-like ATPase with adja-

cent chromodomains that recognize H3K4me3 near the TSS

(reviewed by Persson and Ekwall, 2010). Mammalian Chd1 binds

with higher avidity to H3K4me3 chromatin in vitro. Moreover,

Chd1 is recruited to chromatin via Mediator (Khorosjutina

et al., 2010; Lin et al., 2011), and its transcriptional stimulatory

effect is largely on H3K4me3 chromatin in vitro. Thus, Chd1 is

an H3K4me3 and Mediator-stimulated event, whereas EP400

recruitment is neither, so we did not pursue Chd1’s role in variant

chromatin transcription. In conclusion, our study combines

biochemical and cell-based reporter assays, with genome-

wide analyses. In totality, the data provide a link between H3.3

deposition, EP400 binding and transcription.

EXPERIMENTAL PROCEDURES

Protein Purification

Esa1 and Gcn5 were expressed and purified from E. coli as described (Barrios

et al., 2007). The EP400 cDNA was overexpressed using baculovirus infection

of Sf9 cells for 72h. Sf9 extracts were prepared as described, incubated with

hemagglutinin (HA) beads, and EP400 eluted in 1 mg/ml HA peptide (Lin et al.,

2011).

In Vitro Transcription and IT Assays

The preparation of chromatinizedG5E4T, normalization of chromatin amounts,

in vitro transcription reactions on chromatin templates, immunodepletion, and

IT procedures have been described in our previous publications (Black et al.,

2006; Lin et al., 2011). Briefly, transcription or IT assays were carried out

on 50 ng of chromatin templates that were bound with saturating levels of

GAL4-VP16. Where indicated, immobilized chromatin was acetylated with

20 ng of Esa1, 20 ng Gcn5, and 1 mM acetyl-CoA for 45 min. Transcription

was measured by 32P-primer extension and visualized on an Amersham Bio-

sciences Typhoon 9400. Immunoblots were processed on a LI-COR Odyssey

and quantitated by Image Studio 2. Antibodies included pan-acetyl histone H3

(Active Motif), MED23 (BD PharMingen), Pol II (QED Bioscience), TFIIB (Lin

et al., 2011), EP400, Tip60 (Abcam), ASH2L, CHD1 (Bethyl Laboratories),

and all others from Santa Cruz Biotechnology.

ChIP Analysis in the Tet-VP16 U2OS Cell Line

Stable U2OS Tet-On Luciferase cells were obtained from Clontech Labora-

tories and cultured in Dulbecco’s modified Eagle’s mediumwith 10% Tet-sys-

tem approved fetal bovine serum (Clontech). After addition of 1 mg/ml Dox,

transcription and ChIP assays were as previously described (Black et al.,

2006). Antibodies used in ChIP included MED26 (sc-48776; Santa Cruz

Biotechnology), Pol II (QED Bioscience), H3.3 (H00003021-M01; Abnova),

EP400 (ab70301; Abcam), H2AZ (ab4174; Abcam), and H3K4me1 (ab8895;

MOLCEL 563


Abcam). Antibody against H3K18ac was in house (Ferrari et al., 2008).

Immunoprecipitates were washed, and DNA was quantitated by real-time

semiquantitative PCR. Alternately, mRNA was isolated and quantified by

RT-PCR.

siRNA KD of EP400

The web-based program DEQOR was used to determine unique regions of

EP400 (Henschel et al., 2004) to minimize cross-silencing activities. siRNAs

were produced according to the method described (Fazzio et al., 2008) and

validated by PAGE. Mock siRNA was prepared essentially as described for

EP400 but was from GFP. Cells were treated for 72 hr with either EP400 or

Mock siRNA.

ChIP, Library Preparation, and Analysis

ChIP-seq was performed as described (Ferrari et al., 2012). Libraries were pre-

pared with a KAPA LTP kit using 2 ng DNA, sequenced by the Illumina HiSeq

2000 platform (50 bp reads) andmapped to human genome version hg19 using

bowtie 0.12.9 (Langmead et al., 2009) with 100 bp windows for all proteins

except EP400 (50 bp). Windows with p values < 1.03 10�3 were deemed sig-

nificant peaks. Average ChIP-seq signals, 3 kb upstream and downstream of

the TSS, and metagene plots of average ChIP-seq signals across gene bodies

were calculated by CEAS (Shin et al., 2009). Heatmaps plot p values of enrich-

ment. Significance between Mock and KD was calculated using the Wilcoxon

rank-sum test.

RNA Extraction, mRNA Sequencing Library Preparation, and

Analysis

RNA extraction, library preparation, and sequencing analysis were performed

essentially as described (Ferrari et al., 2012). Libraries were sequenced as

above to obtain single end 50 bp-long reads. Reads were aligned as for

ChIP but using tophat 2.0.8 (Trapnell et al., 2009). FPKM values were deter-

mined using Cuffdiff 2.0.2 (Trapnell et al., 2010).

In Vitro Histone Exchange Assays

Histone exchange assays were performed as described (Mizuguchi et al.,

2004). One hundred fifty nanograms of immobilized G5E4T DNA or 300 ng of

immobilized canonical chromatin was incubated in 100 ml with 40 ng recombi-

nant EP400 and 300 ng of H3.3-H4 tetramers, or H2AZ-H2B dimers with 1 mM

ATP for 1 hr at 37�C. Chromatin was captured on beads andwashed twicewith

exchange buffer (0.4 M KCl) and once with buffer containing 70 mM KCl.

Bound proteins were eluted with SDS-PAGE loading buffer. The washes and

supernatants were combined, TCA precipitated, and fractionated on SDS

gels alongside bound fractions, and immunoblotted.

ACCESSION NUMBERS

The accession number for the genomics data reported in this paper is GEO:

GSE73742.

AUTHOR CONTRIBUTIONS

S.K.P. andM.F.C. conceived and designed the experiments. S.K.P. performed

the experiments. S.K.P. and T.S. performed informatics analyses. C.H. deter-

minedMED26ChIP conditions. L.Y., J.C., and K.J. performed cloning, expres-

sion, and purification of recombinant proteins. J.C. and K.C. provided critical

reagents and technical advice. S.K.P. and M.F.C. wrote the manuscript with

input from J.C. and S.K.K.

ACKNOWLEDGMENTS

This work was supported by Canadian Institutes of Health Research grant

MOP-64289 to J.C. and National Institutes of Health grants GM074701 to

M.F.C. and CA178415 to S.K.K. L.Y. was supported by a Ruth L. Kirschstein

National Research Service Award (GM007185). We thank Kostas Chronis for

discussions and technical assistance and Swami Venkatesh for manuscript

comments.

9


Received: July 9, 2015

Revised: September 28, 2015

Accepted: October 26, 2015

Published: December 3, 2015

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ep400 deposits h3.3 into promoters and enhancers during ......transformation (fuchs et al., 2001)....

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