The authors have declared that no competing interests exist.
Conceived and designed the experiments: RD MKN ES PC. Performed the experiments: RD MKN ES TK. Analyzed the data: RD MKN ES VI TK PC. Contributed reagents/materials/analysis tools: VI TK. Wrote the paper: RD PC.
Current address: Department of Biology, York University, Toronto, Canada
H2A.Z is an essential histone variant that has been implicated to have multiple chromosomal functions. To understand how H2A.Z participates in such diverse activities, we sought to identify downstream effector proteins that are recruited to chromatin via H2A.Z. For this purpose, we developed a nucleosome purification method to isolate H2A.Z-containing nucleosomes from human cells and used mass spectrometry to identify the co-purified nuclear proteins. Through stringent filtering, we identified the top 21 candidates, many of which have conserved structural motifs that bind post-translationally modified histones. We further validated the biological significance of one such candidate, Brd2, which is a double-bromodomain-containing protein known to function in transcriptional activation. We found that Brd2's preference for H2A.Z nucleosomes is mediated through a combination of hyperacetylated H4 on these nucleosomes, as well as additional features on H2A.Z itself. In addition, comparison of nucleosomes containing either H2A.Z-1 or H2A.Z-2 isoforms showed that significantly more Brd2 co-purifies with the former, suggesting these two isoforms engage different downstream effector proteins. Consistent with these biochemical analyses, we found that Brd2 is recruited to AR–regulated genes in an H2A.Z-dependent manner and that chemical inhibition of Brd2 recruitment greatly inhibits AR–regulated gene expression. Taken together, we propose that Brd2 is a key downstream mediator that links H2A.Z and transcriptional activation of AR–regulated genes. Moreover, this study validates the approach of using proteomics to identify nucleosome-interacting proteins in order to elucidate downstream mechanistic functions associated with the histone variant H2A.Z.
Within the cell's nucleus, DNA closely associates with histone proteins, forming a structure known as chromatin. Packaging DNA into chromatin allows for efficient storage of the genome, and it also provides an additional means of regulating processes, such as gene expression, that require access to DNA. Two copies each of the four core histones (H2A, H2B, H3, H4) associate with approximately 150 base pairs of DNA to make up the basic unit of chromatin, the nucleosome. In addition to the core histones, variants exist that have specialized functions within chromatin. One such variant is H2A.Z, which is essential for cell viability. Here, we describe an approach by which to characterize proteins that interact with H2A.Z-containing nucleosomes. Our findings reveal that many of the identified proteins may interact with H2A.Z nucleosomes by recognizing specific chemical modifications uniquely present on H2A.Z nucleosomes. One such protein, Brd2, interacted in a manner dependent on recognition of acetylated histone residues that are enriched on H2A.Z nucleosomes. Furthermore, this interaction is required for expression of hormone-responsive genes in prostate cancer cells. By this approach, we uncovered a key mediator linking H2A.Z to transcriptional regulation and found a potentially targetable step to regulate prostate cell proliferation.
H2A.Z is a variant of the canonical histone H2A. Amongst the different variants of core histones that have been identified to date, H2A.Z is unique in being the only variant that is essential for viability and development in a number of organisms
At the amino acid level, mammalian H2A and H2A.Z share about 60% identity. Knockout of the H2A.Z gene is lethal in mice, which suggests that the unique regions of H2A.Z are required for the essential function in complex eukaryotes. Moreover, these unique regions likely engage effector proteins that mediate H2A.Z-specific functions. A number of studies have examined and identified H2A.Z-interacting proteins; however, these studies have focused on purifying soluble, tagged H2A.Z, often from whole-cell extracts, to identify proteins interacting directly with this variant
Using our nucleosome purification-mass spectrometry analyses, we indeed identified a number of proteins that preferentially associate with H2A.Z-nucleosomes over H2A-nucleosomes. Gene ontology (GO) analyses showed that the majority of the interacting proteins are chromosome- or chromatin-associated proteins. Consistent with the transcription-related functions of H2A.Z, many of the identified proteins have putative transcription-associated functions. Of the top 21 identified proteins, we focused our follow-up studies on Brd2 because of its transcriptional co-activator function and because it contains bromodomains that bind acetyl-lysines.
Brd2 belongs to the BET family of proteins, all of which contain tandem bromodomains in their N-termini and an extraterminal domain of unknown function in their C-termini
In this study, we found that Brd2's association with H2A.Z nucleosomes is enhanced upon treatment of cells with trichostatin A (TSA) and is dependent on its bromodomains. Peptide competition assays suggest that acetylated H4 is the primary site of interaction between Brd2 and H2A.Z nucleosome and, indeed, we found that the overall H4 acetylation levels are higher on these nucleosomes than on H2A nucleosomes. However, experiments using re-assembled H2A- and H2A.Z-nucleosomes that contain equivalent amounts of H4 acetylation suggest that additional features specific to H2A.Z further augment Brd2's interaction with nucleosomes. This conclusion is further supported by the fact that more Brd2 co-purifies with H2A.Z-1, compared to H2A.Z-2, -containing nucleosomes, even though they both have similar levels of H4 acetylation.
To understand the physiological functions of chromosomal H2A.Z, we took an unbiased proteomics approach to identify proteins preferentially interacting with H2A.Z-containing nucleosomes. To do this, we first transfected 293T cells with Flag-tagged H2A.Z (or Flag-tagged H2A as a control), digested the chromatin to mononucleosomes with micrococcal nuclease (MNase) and then immunoprecipitated intact nucleosomes with anti-Flag antibody. By this method, we isolated and analyzed the co-purifying proteins by LC-MS/MS (
A. Schematic of the nucleosome IP & mass spectrometry protocol used. B. Heat map of spectral counts from amalgamated data showing the top 21 protein hits. C. Venn diagram summarizing the number of unique and overlapping hits between H2A.Z nucleosomes, H2A nucleosomes, and the Flag-tagged GFP control. D. Gene ontology analysis of the proteins identified in our MS analysis.
Interestingly, many of the other identified proteins have conserved domains that can recognize various histone PTMs. For example, PHF6 and PHF14 have PHD fingers, which are motifs that can recognize methylated lysine residues
Brd2 is a double-bromodomain protein known to be involved in transcriptional activation. Given that both H2A.Z and Brd2 are known to have transcription-related functions, we chose to perform follow-up experiments to further validate and characterize the interaction between Brd2 and H2A.Z nucleosomes. First, we confirmed this interaction by repeating our mononucleosome IP experiments and compared the amounts of endogenous Brd2 that co-immunoprecipitated with the H2A/H2A.Z nucleosomes by Western blots (
A. H2A.Z and H2A nucleosomes were purified as described in
To determine if the enhancement of Brd2 binding to H2A.Z nucleosomes following hyperacetylation is specific to Brd2, we compared the binding properties of Brd2 to that of a known H2A.Z-binding protein, VPS72. VPS72 is the human homologue of Swc2, which is a component of the H2A.Z deposition complex Swr1, and directly interacts with H2A.Z, within the Swr1 complex
Since the interaction between Brd2 and H2A.Z nucleosomes greatly increased when the chromatin is hyperacetylated, this suggests that the interaction is mediated through the bromodomains of Brd2 and the acetylated lysines on histones. To test this, we compared the ability of H2A.Z nucleosomes to immunoprecipitate wild type (WT) Brd2, to a mutant version (BD) of Brd2 that contains point mutations in each of its bromodomains, which renders the domains incapable of binding acetylated lysine residues
Previously, it has been reported that the bromodomains of Brd2 bind acetylated lysine residues on H4
A. A schematic depicting the experimental design of the peptide competition assay. B. Western blots of eluted material from H2A.Z mononucleosome IPs performed in the presence of various competing peptides—sites of acetylation of the various peptides are indicated (UN, unacetylated peptide). H4 peptides acetylated at K12 alone or at K5, K8, K12 and K16 (Tetra) were able to efficiently compete away Brd2 binding to H2A.Z nucleosomes. C. H2A and H2A.Z nucleosomes were immunoprecipitated following treatment with DMSO or TSA—eluted material was analyzed by Western blot for various acetylation marks on H4 and H3. H2A.Z nucleosomes contain higher levels of acetylated H3 and H4 under both basal and hyperacetylated conditions. AcLys, an anti-pan-acetyl lysine antibody, which detects both acetyl H4 and H3 bands in our Western blot, was used as indicated.
In light of this finding, we examined the acetylation levels on H2A.Z and H2A nucleosomes. We immunoprecipited either H2A.Z- or H2A-nucleosomes and we compared the levels of acetylated H4 and H3 residues by Western blot. As shown in
To directly test this possibility, we generated H2A- and H2A.Z-nucleosomes that have equivalent amounts of H4 acetylation and asked whether Brd2 binds differentially to these modified nucleosomes (
A. Schematic workflow of nucleosome preparation used to randomly re-assemble H2A–H2B and H2A.Z-H2B dimers with H3–H4 tetramers, generating H2A- and H2A.Z-nucleosomes with comparable levels of H3 and H4 PTMs. B. Western blot comparison of “scrambled” versus “non-scrambled” nucleosomes for H3 and H4 PMTs and Brd2 binding. Immunoprecipitated nucleosomes subjected to re-assembly via high-salt/low-salt dialysis (scrambled) show comparable levels of H4 acetylation and H3 methylation compared to non-scrambled control nucleosomes, which show an enrichment of these marks on H2A.Z nucleosomes. Consistent with previous experiments, Brd2 shows preferential enrichment on H2A.Z nucleosomes in the non-scrambled control samples. Brd2 still shows a slight preference for H2A.Z nucleosomes, compared to H2A nucleosomes even when levels of H4 acetylation are comparable in the scrambled nucleosomes. The dashed lined between the two Flag blots is to indicate that a single membrane was cut and therefore Flag-NLS-GFP and Flag-H2A/H2A.Z blots are shown as separate panels. C. Comparison of Brd2 interaction with mononucleosomes containing the different isoforms of H2A.Z. Mononucleosomes IPs were performed as described in
Recent studies found that there are in fact two isoforms of H2A.Z (H2A.Z-1 and H2A.Z-2) that differ by 3 amino acids
To test the biological significance of the interaction between H2A.Z nucleosomes and Brd2, we examined their relationship in the context of androgen receptor (AR)- regulated genes since we and others have previously reported that H2A.Z is required for the full activation of AR-regulated genes in the prostate cancer cell line LNCaP
A. A schematic of the prostate specific antigen (PSA) gene. Arrows indicate approximate positions of primers used for qPCR analysis. “(−)2 Kb” represents the region in between the enhancer and promoter and was used as a negative control region, which is approximately 2 kb upstream of the transcription start site. B. ChIP analysis of the PSA gene following a time-course of androgen stimulation (10 nM)—antibodies used for immunoprecipitation are shown on the left. H2A.Z and H4Ac (tetra-acetylated H4) ChIP data were normalized to total H3 signal (data not shown) to account for changes in nucleosome density and are therefore presented as “H2A.Z/H4Ac enrichment”. Each qPCR reaction was performed in triplicate with each experiment repeated at least three times independently. Values are presented as means, ± standard deviation.
As we discovered that Brd2 is recruited to AR-regulated genes upon hormone activation, we next asked whether this recruitment is dependent on H2A.Z. To that end, we examined Brd2 recruitment by ChIP analysis in the H2A.Z knockdown cells that we have previously generated
A. Western blot analyses of whole-cell lysates from LNCaP cells stably expressing either a scrambled control shRNA or an shRNA targeting H2A.Z-1 mRNA. H2A.Z knockdown does not significantly affect the protein levels of AR or Brd2, nor does it reduce global levels of tetra-acetylated H4 (H4Ac). Tubulin and H3 are shown for the purpose of loading controls. B. ChIP analysis of Brd2 and tetra-acetylated H4 at the PSA and KLK2 genes. Knockdown of H2A.Z reduces the recruitment of Brd2 as well as the acetylation of H4 at AR-regulated genes following hormone stimulation for 60 minutes. The data represent the fold-enrichment in hormone-stimulated cells relative to respective ethanol-treated controls (vehicle control). H4Ac ChIP was also normalized to H3 to account for changes in nucleosome density. Each qPCR reaction was performed in triplicate with each experiment repeated at least three times independently. Values are presented as means, ± standard deviation.
The recruitment of Brd2 to AR-regulated genes has not been reported before. Moreover, we discovered that the recruitment is dependent on the presence of H2A.Z. To test the importance of Brd2 in the transcriptional activation of AR-regulated genes, we initially attempted to generate stable Brd2 knockdown LNCaP cells using Brd2-targeting shRNA constructs. However, cells that showed good knockdown of Brd2 also displayed growth defects, which confounded the AR-activation studies. Brd2 is an essential gene and published literature has alluded to the fact that studying Brd2 function by shRNA knockdown is problematic
A. H2A.Z mononucleosome IPs were performed using cells pre-treated with DMSO or the small molecule inhibitor JQ1. Starting input material (prior to adding beads) and eluted material (post IP) was analyzed by Western blotting. Both concentrations of JQ1 (125 nM and 250 nM) were able to reduce the amount of Brd2 immunoprecipitating with H2A.Z nucleosomes. A slight reduction of Brd2 in the INPUT fraction is also observed following JQ1 treatment, as well as a slight reduction in the levels of H4Ac in the IP fraction (see main text for discussion). H3 and Flag blots are shown for loading purposes. B. Whole-cell lysates of LNCaP cells treated with JQ1 (125 nM or 250 nM) or DMSO were analyzed by Western blot. JQ1 treatment does not reduce total levels of Brd2, AR or H2A.Z although a small decrease in total levels of H4Ac are observed (see main text). Alpha tubulin and H3 are shown as loading controls. C. PSA and KLK2 regulatory regions were analyzed by ChIP for Brd2 recruitment and H4 acetylation (tetra-acetylated) in cells stimulated with ethanol or DHT (10 nM) for 60 minutes. JQ1 treatment reduces the recruitment of Brd2 to both the enhancer and promoter regions of PSA and KLK2 and reduces the H4 acetylation levels to a lesser extent. The data represent the fold-enrichment in hormone-stimulated cells relative to respective ethanol-treated controls (vehicle control). H4Ac ChIP was also normalized to H3 to account for changes in nucleosome density. Each qPCR reaction was performed in triplicate with each experiment repeated at least three times independently. Values are presented as means, ± standard deviation. D. RT-qPCR analysis of LNCaP cells pre-treated with JQ1 or DMSO for 24 hrs then stimulated with 10 nM of androgen for 24 hrs. Analysis of both the PSA and KLK2 genes showed a dose-dependent decrease in expression in cells pre-treated with JQ1. E. MTS proliferation assay of LNCaP cells treated with various concentration of JQ1, or an equivalent volume of DMSO, for 24 hrs. Absorbance values were normalized to non-treated control wells and presented as “Proliferation Index”. Assay wells were prepared in triplicate and the experiment was repeated three times independently. Values are presented as means, ± standard deviation.
Given that JQ1 disrupts the interaction between Brd2 and H2A.Z nucleosomes, we next tested whether JQ1 also blocks recruitment of Brd2 to the AR-regulated genes. Analysis of whole-cell lysates from LNCaP cells showed that JQ1 treatment does not affect total levels of Brd2, AR, or H2A.Z, and only causes a slight reduction in total levels of acetyl H4 (
In order to gain a deeper understanding of the physiological functions of H2A.Z, we reasoned that proteins acting downstream of H2A.Z must first engage this histone variant in the context of chromatin. Therefore, we specifically chose to perform a proteomics screen to identify proteins that preferentially associate with H2A.Z nucleosomes, as compared to nucleosomes containing the core histone H2A. This is a departure from previously published approaches that focused on identifying and characterizing soluble H2A.Z-interacting proteins, which led to the identification of histone chaperones and remodeling complexes, such as the Swr1 complex, which deposits H2A.Z into chromatin
In support of this multivalency model, we found that the binding of Brd2 to H2A.Z-nucleosomes is primarily mediated through the interactions between the bromodomains on Brd2 and the high levels of H4 acetylation on H2A.Z nucleosomes. In addition, there are likely other non-H4-acetylation-dependent contact points. This conclusion is based on two separate lines of evidence: First, using high salt/low salt dialysis to re-assemble nucleosomes that have randomly mixed histone compositions, we found that even though these H2A/H2A.Z nucleosomes now have almost equivalent amounts of H4 acetylation, Brd2 still shows a small but distinct preference for H2A.Z-containing nucleosomes. Second, significantly more Brd2 co-purified with H2A.Z-1 nucleosomes, compared to H2A.Z-2 nucleosomes, even though both types of nucleosomes have similar levels of H4 acetylation. Therefore, these findings strongly suggest that additional features or surfaces on H2A.Z (H2A.Z-1 in particular) provide further interaction sites to stabilize the binding of Brd2 to H2A.Z nucleosomes.
At present, we have not characterized, nor identified, the exact determinants that result in the differential binding of Brd2 to the H2A.Z-1 and H2A.Z-2 nucleosomes. This finding is intriguing given that the two isoforms differ only by 3 amino acids. Nevertheless, it is not without precedence since H3.3 and H3.1 only differ by 4 amino acids and yet they are physically associated with distinct chaperone complexes
In addition to characterizing Brd2 as an H2A.Z nucleosome-binding protein, we also demonstrated that Brd2 is a novel regulator of androgen responsive genes in LNCaP cells. Our ChIP analyses of the PSA gene in LNCaP cells showed that Brd2 is recruited to the promoter and enhancer regions following stimulation of cells with hormone. More importantly, we found that recruitment of Brd2 is dependent on H2A.Z since knockdown of H2A.Z resulted in reduced levels of Brd2 recruitment, as well as H4 acetylation levels, at the enhancers and promoters of PSA and KLK2 genes. We note that the knockdown construct we used is designed to target H2A.Z-1, and it is unlikely that this shRNA also targets H2A.Z-2 since the DNA sequences of the two genes are quite divergent. Nevertheless, given that Brd2 preferentially associates with H2A.Z-1 nucleosomes, the effects seen with just knocking down H2A.Z-1 is not surprising. Currently, there are no antibodies, nor shRNAs, developed that are specific for the H2A.Z-2 isoform. Once such reagents are available, it would be interesting to test whether H2A.Z-2 has a functional role for the expression of AR-regulated genes.
Prior to this study, the role of Brd2 in AR-regulated gene expression had not been reported. The importance of Brd2, and possibly other BET proteins, in this process is supported by our studies using the small molecule inhibitor JQ1. JQ1 treatment not only disrupted binding of Brd2 to H2A.Z nucleosomes, but also strongly affected the expression of AR-regulated genes and proliferation of LNCaP cells. We note that JQ1 inhibits other BET family members as well
Our data all together led us to propose a model whereby, at AR-regulated genes, H2A.Z establishes a unique platform for the recruitment of transcriptional co-activators, such as Brd2 (see
Following hormone stimulation, Brd2 is recruited to H2A.Z nucleosomes containing acetylated H4 lysines. Association of Brd2 with histone acetyltransferase (HAT) activity promotes acetylation of neighboring nucleosomes. Recruitment of chromatin remodeling activity causes eviction of H2A.Z nucleosomes, which promotes the recruitment of DNA-binding transcription factors, such as AR, and spreading of the acetylated H4 mark promotes a subsequent spreading of Brd2.
In conclusion, our study has provided novel insights into the physiological functions of H2A.Z and its ability to engage chromatin-binding proteins through its influence on PTMs within the nucleosome. Our characterization of the interaction between Brd2 and H2A.Z nucleosomes also furthered our understanding of the role H2A.Z plays in promoting AR-regulated transcription in prostate cancer cells, yielding potential new molecular targets for therapy. The data generated from our MS analysis of proteins binding to H2A.Z nucleosomes will serve as a useful tool in future studies of H2A.Z's role in various chromatin-templated processes.
293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. LNCaP cells were obtained from ATCC and were grown in RPMI 1640 media supplemented with 10% fetal bovine serum. For culturing in the absence of hormone, cells were grown in phenol-red free RPMI 1640 supplemented with 5% charcoal-stripped fetal bovine serum (Invitrogen) for 72 hrs prior to treatment with hormone. Dihydrotestosterone (DHT) was obtained from Sigma and re-suspended in absolute ethanol; DHT was added to cells at a final concentration of 10 nM, or for control samples, an equivalent volume of ethanol was added. For treatment of cells with trichostatin A (TSA), cells were treated with TSA (200 nM) or an equivalent volume of DMSO for 2 hours prior to harvest. The JQ1 reagent was kindly provided by Dr. Jay Bradner, Dana-Farber Cancer Institute. All transfections were carried out using Lipofectamine 2000 (Invitrogen). All expression constructs used were based on the pcDNA 3.1 (+) (Invitrogen) backbone with the Flag tag cloned in-frame. H2A.Z antibody directed against the L1 loop was described previously
Generation of mononucleosomes was performed as described previously
Raw data was converted to m/z XML using ReAdW and searched by X!Tandem against a locally installed version of the human UniProt complete human proteome protein sequence database (release date 2009, 20,323 sequences). The search was performed with a fragment ion mass tolerance of 0.4 Da and a parent ion mass tolerance of ±10 ppm. Complete tryptic digest was assumed. Carbamidomethylation of cysteine was specified as fixed and oxidation of methionine as a variable modification. A target/decoy search was performed to experimentally estimate the false positive rate and only proteins identified with two unique high quality peptide identifications were considered as previously reported
IPs were performed essentially as described above, with the following modification: Peptides were added to the input material giving a final peptide concentration of 30 µg/ml, then incubated at 4°C with rotation for 30 min. M2-agarose beads were then added as described above.
293T cells were transfected with a construct expressing Flag-H2A, Flag-H2A.Z, or GFP-NLS, as described previously. Mononucleosomes were prepared as described above, and dialyzed against 1.2 M NaCl, 10 mM Tris pH 8.0, 0.2 mM EDTA, 1 mM DTT, 5 mM sodium butyrate overnight at 4°C. Nucleosomes were then reconstituted in the same buffer by salt-gradient dialysis at 4°C as follows: 0.9 M NaCl, 2 h; 0.6 M NaCl, 2 h; 0.3 M NaCl, 2 h. The final dialysis was for 3 h against 140 mM NaCl, 20 mM Tris pH 7.6, 5 mM sodium butyrate. Flag immunoprecipitation was carried out as described. Following washes, nucleosomes bound to Flag beads were subsequently incubated overnight at 4°C with salt-extracted nuclear lysates prepared as described in
Cells stably expressing the H2A.Z (cagctgtccagtgttggtg) shRNA target sequence were generated as described previously
RT-qPCR or ChIP analysis of LNCaP cells was performed as previously described
LNCaP cells were seeded into 96-well tissue culture plates and grown for 24 hrs as described. Cells were treated with various concentrations of JQ1, or an equivalent volume of DMSO, for 24 hrs, followed by the addition of CellTitre 96 AQueous One Solution reagent (Promega) according to manufacturer's instructions. Absorbance was measured at 490 nM using a Synergy H4 Hybrid Microplate Reader (BioTek).
Validation of H2A.Z nucleosome-interacting proteins identified by mass spec. Two proteins identified in our mass spec analysis, USP39 (A) and PWWP2A (B), were validated by generating HA-tagged expression constructs of each and co-transfecting the construct with either Flag-H2A, Flag-H2A.Z, or Flag-NLS-GFP. Mononucleosomes were harvested from the transfected cells as described in
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Validation of the interaction between Brd2 and H2A.Z nucleosomes in LNCaP cells. Mononucleosome IPs were performed as described in
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AR ChIP in LNCaP Cells. ChIP was performed using chromatin from LNCaP cells as described in
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Brd4 interacts with H2A.Z nucleosomes and is recruited to the PSA gene in a manner that is inhibited by JQ1. A. Mononucleosomes were isolated from cells expressing either Flag-H2A or Flag-H2A.Z, and treated with TSA or vehicle control (DMSO)—see
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