Skip to main content
NCBI home page
iScience logoLink to iScience
. 2019 Oct 31;21:773–788. doi: 10.1016/j.isci.2019.10.053

A Read/Write Mechanism Connects p300 Bromodomain Function to H2A.Z Acetylation

Yolanda Colino-Sanguino 1,2,3,7, Evan M Cornett 4,7, David Moulder 1,2,3, Grady C Smith 2,3, Joel Hrit 5, Eric Cordeiro-Spinetti 5, Robert M Vaughan 5, Krzysztof Krajewski 6, Scott B Rothbart 5,8,∗, Susan J Clark 2,3,8,∗∗, Fátima Valdés-Mora 1,2,3,8,9,∗∗∗
PMCID: PMC6889796  PMID: 31727574

Summary

Acetylation of the histone variant H2A.Z (H2A.Zac) occurs at active regulatory regions associated with gene expression. Although the Tip60 complex is proposed to acetylate H2A.Z, functional studies suggest additional enzymes are involved. Here, we show that p300 acetylates H2A.Z at multiple lysines. In contrast, we found that although Tip60 does not efficiently acetylate H2A.Z in vitro, genetic inhibition of Tip60 reduces H2A.Zac in cells. Importantly, we found that interaction between the p300-bromodomain and H4 acetylation (H4ac) enhances p300-driven H2A.Zac. Indeed, H2A.Zac and H4ac show high genomic overlap, especially at active promoters. We also reveal unique chromatin features and transcriptional states at enhancers correlating with co-occurrence or exclusivity of H4ac and H2A.Zac. We propose that differential H4 and H2A.Z acetylation signatures can also define the enhancer state. In conclusion, we show both Tip60 and p300 contribute to H2A.Zac and reveal molecular mechanisms of writer/reader crosstalk between H2A.Z and H4 acetylation through p300.

Subject Areas: Biological Sciences, Biochemistry, Molecular Biology, Cell Biology

Graphical Abstract

graphic file with name fx1.jpg

Open in a new tab

Highlights

  • •

    p300 acetylates H2A.Z at multiple N-terminal lysine residues

  • •

    Interaction of p300 bromodomain with H4ac enhances H2A.Zac

  • •

    H2A.Zac and H4ac co-localize at active regulatory regions

  • •

    H4ac and H2A.Zac differential signature can define the enhancer state

Biological Sciences; Biochemistry; Molecular Biology; Cell Biology

Introduction

Histone variants replace core histones to perform a variety of specialized functions (Kamakaka and Biggins, 2005) and, as canonical histones, are prone to posttranslational modifications (PTMs). H2A.Z is an evolutionarily conserved histone variant of the canonical histone H2A, which shares ∼60% identity in amino acid sequence (reviewed in Zlatanova and Thakar, 2008). H2A.Z has been implicated in many diverse and potentially opposing functions, including regulation of gene transcription, where H2A.Z has been associated with active, poised, or inactive gene expression. The contrasting roles of H2A.Z are associated with different PTMs, including lysine acetylation, monoubiquitination, and methylation (reviewed in Sevilla and Binda, 2014) (Colino-Sanguino et al., 2016).

Acetylated H2A.Z (H2A.Zac) is associated with active transcription (Bruce et al., 2005, Halley et al., 2010, Millar et al., 2006, Valdes-Mora et al., 2012) and promotes nucleosome destabilization and an open chromatin conformation (Ishibashi et al., 2009). In mammals, lysines 4, 7, and 11 (K4, K7, K11) in the H2A.Z N-terminal tail are the most frequently acetylated residues (Ishibashi et al., 2009). Genome-wide studies based on H2A.Zac ChIP on-chip or ChIP-seq in different cell types and organisms have revealed that the acetylated H2A.Z forms are primarily restricted to genomic regulatory regions, such as promoters and enhancers (Bruce et al., 2005, Brunelle et al., 2015, Hu et al., 2013, Ku et al., 2012, Millar et al., 2006, Valdes-Mora et al., 2017, Valdes-Mora et al., 2012). We and others have shown that H2A.Zac is associated with aberrant gene expression in prostate (Dryhurst et al., 2012, Valdes-Mora et al., 2012) and breast cancer (Bellucci et al., 2013, Dalvai et al., 2012, Dalvai et al., 2013). Recently, we found that increased H2A.Zac levels correlate with poor prognosis in prostate cancer patients and demonstrated a pro-oncogenic role for H2A.Zac through the ectopic activation of cancer-related enhancers (Valdes-Mora et al., 2017). Collectively, these data suggest that inhibition of H2A.Z acetylation may be a therapeutic strategy for treating prostate cancer. However, the molecular mechanisms and key players responsible for the acetylation of H2A.Z are not fully understood.

Histone lysine acetylation is catalyzed by lysine acetyltransferases (KATs). In mammalian cells, nine nuclear KATs have been identified, from which three—Tip60, CREB binding protein (CBP), and E1A binding protein p300 (p300) —can acetylate canonical histones (Ito et al., 2000, Jeong et al., 2011). In the budding yeast Saccharomyces cerevisiae, the lysine acetyltransferase NuA4, considered the orthologue of Tip60 (Doyon et al., 2004), is reported to acetylate Htz1, the yeast orthologue of H2A.Z (Altaf et al., 2010, Keogh et al., 2006, Millar et al., 2006). Indeed, studies in mammalian cells have suggested a link between H2A.Zac and the Tip60 complex in locus-specific experiments (Dalvai et al., 2012, Dalvai et al., 2013, Giaimo et al., 2018) and by immunoprecipitation of the Tip60 complex followed by KAT assays with recombinant H2A.Z (Ito et al., 2018). However, other studies showed that H2A.Zac was independent of Tip60 inhibition at locus-specific sites (Bellucci et al., 2013, Narkaj et al., 2018) and in the context of DNA repair (Semer et al., 2019), suggesting that other KATs may also be responsible for H2A.Zac.

CBP and p300 (also known as CREBBP or KAT3A and EP300 or KAT3B, respectively) are highly homologous proteins that comprise a unique KAT family and are often referred to interchangeably. p300/CBP is a transcriptional co-activator and catalyses lysine 27 acetylation of histone H3 (H3K27ac) at promoters and enhancers (Creyghton et al., 2010, Hilton et al., 2015). p300/CBP is a multifunctional protein that acts as both a “writer” and a “reader” of lysine acetylation through its KAT domain and bromodomain (BD), respectively (Dancy and Cole, 2015, Zeng et al., 2008). p300/CBP is a promiscuous KAT that can acetylate all four canonical histones (Weinert et al., 2018). However, p300/CBP's ability to acetylate and/or interact with H2A.Z has not been critically evaluated.

In this study, we have interrogated two of the most likely KAT candidates for the acetylation of H2A.Zac: Tip60 and p300/CBP. Here, we provide in vitro evidence, with peptide and recombinant nucleosome substrates, that Tip60 does not efficiently acetylate H2A.Z. In contrast, we demonstrate that p300 can acetylate H2A.Z at multiple lysine residues, both in vitro and in human cells, and H2A.Zac is enhanced by p300 BD-mediated H4ac reader activity. In support of this mechanism, we find a high degree of genomic overlap between H2A.Zac and H4ac at active regulatory regions, preferentially at active promoters. However, at enhancers, we find that H2A.Zac and H4ac nucleosome occupancy is differentially associated with distinct chromatin features and the transcriptional activity of the genomic region. Overall, our findings suggest that in addition to Tip60, p300/CBP is also required for H2A.Z acetylation, providing new insights for the modulation of H2A.Zac pro-oncogenic activity in prostate cancer.

Results

p300 Acetylates H2A.Z In Vitro

To test whether our putative KAT candidates acetylated H2A.Z, we performed in vitro lysine acetyltransferase assays with recombinant Tip60 and p300 (Figures S1A and S1B). As substrates, we used biotinylated peptides corresponding to the first 19 amino acids of H2A.Z (Figure 1A) and observed acetylation rates by measuring radioactive acetyl group incorporation (Figure 1B). As a negative control, we used an H2A.Z peptide containing the most commonly acetylated lysines (K4, K7, and K11) (Hu et al., 2013, Ishibashi et al., 2009). For positive controls, known substrates for each KAT were used, including an H4 N-terminal peptide for Tip60 (Kimura and Horikoshi, 1998) and an H3 peptide flanking H3K27 for p300 (Jin et al., 2011). Recombinant Tip60 rapidly acetylated the positive control H4 peptide. However, H2A.Z only showed a slight increase in acetylation signal compared with the negative control H2A.ZK4acK7acK11ac (Figure 1B, upper panel and Figure S1C). These data suggest that H2A.Z peptides are not optimal substrates for recombinant Tip60. In contrast, recombinant p300 (catalytic domain plus BD) (Figure S1A), which shares 82% of protein domain identity with CBP, acetylated H2A.Z peptides at a similar rate to its most appreciated histone substrate, H3K27 (Figure 1B, bottom panel). We confirmed this activity with full-length recombinant p300 (Figure S1D). p300 activity toward peptide substrates of H2A.Z isoform 2 (Dryhurst et al., 2009) was similar to isoform 1 (referred herein as H2A.Z) (Figures S1E and S1F).

Figure 1.

Figure 1

Open in a new tab

H2A.Z Is a Substrate for p300 In Vitro Assays

(A) H2A.Z-1 N-terminal amino acid sequence for the peptides used in panel (B). All lysines that can be acetylated are shown in red.

(B) In-solution H3-Acetyl-CoA (AcCoA) assays measuring Tip60 (top) and p300 (bottom) activity as a function of time on the following histone peptide substrates: un-acetylated H2A.Z (peptide spans from amino acid 1–19, H2A.Z-1 (1-19)) and the tri-acetylated H2AZ at lysines 4, 7, and 11 (1–19) (H2A.Z-1 (1-19)K4acK7acK11ac) were used for both Tip60 and p300 assays. As controls we used H4 (1–23) and acetylated H4 at lysines 5, 8, 12, and 16 (1–23) (H4(1-23)K5acK8acK12acK16ac) for Tip60 and H3 (15–34) and acetylated H3 at lysine 27 peptides (15–34) (H3(15-34)K27ac) for p300. Data points are presented as mean count per minute (cpm). Error bars represent the standard deviation (SD) from two measurements.

(C) Coomassie staining (left) and H3 fluorography (right) of recombinant canonical nucleosomes (canonical nuc) and homotypic H2A.Z-1 nucleosomes (H2A.Z-1 nuc) incubated with Tip60 in the presence or absence of H3-AcCoA for 12 h. White bands in the autoradiography are the overlayed molecular weight markers shown in the Coomassie staining. Representative image of two replicates.

(D) H3 fluorography (top) and Coomassie staining (bottom) of recombinant canonical nucleosomes (canonical nuc) and homotypic H2A.Z-1 and H2A.Z-2 nucleosomes (H2A.Z-1/H2A.Z-2 nuc) incubated with p300 in the presence or absence of H3-AcCoA for 30 min. Representative image of two replicates.

(E) In-solution H3-AcCoA KAT assays measuring p300 activity as a function of time on nucleosome substrates, as indicated. Data points are presented as mean count per minute (cpm). Error bars represent the SD from two measurements.

(F) Percentage of area under the mass spectrometry (MS) peak of unacetylated, one acetylated lysine (mono-ac), two acetylated lysines (di-ac), three acetylated lysines (tri-ac), or four acetylated lysines (tetra-ac) from H2A.Z peptides at increasing p300 incubation time points, 0, 4, 8, 16, 32, and 64 min (raw data are displayed in Figure S2.). Data are represented as mean −/+ SD of two independent replicates.

See also Figures S1 and S2.

H2A.Z-containing nucleosomes have slightly different biophysical properties than those containing canonical H2A, including an extended acidic path on the nucleosome surface (Suto et al., 2000), which may affect the interactions with KAT domains. To confirm our peptide results on more physiologically relevant substrates, we performed in vitro KAT assays using recombinant mononucleosome substrates (Figures 1C and 1D). Reactions were separated by gel electrophoresis and incorporation of tritiated acetyl groups was detected using autoradiography, allowing deconvolution of which histones were acetylated. Consistent with our peptide results, recombinant Tip60 acetylated H4 in both canonical and H2A.Z-1-containing nucleosomes (Figure 1C); however, no acetylation of H2A.Z was detected. Moreover, we found Tip60 autoacetylation, which positively regulates Tip60 catalytic activity (Yang et al., 2012), suggesting that the recombinant Tip60 is functionally active. In contrast, p300 acetylated all histones in the canonical nucleosomes after 30 min of incubation (Figure 1D), as previously reported (Ogryzko et al., 1996, Schiltz et al., 1999). Notably, p300 also acetylated all histones in the H2A.Z-1- and H2A.Z-2-containing nucleosomes, including both H2A.Z isoforms (Figure 1D). Canonical nucleosomes were a slightly better substrate than variant-containing nucleosomes, but no differences were found in the acetylation rates between nucleosomes containing H2A.Z-1 and H2A.Z-2 isoforms (Figure 1E).

Mass spectrometry data from human cells have shown that H2A.Zac is found most commonly as a tri-acetylated form, with lysines 4, 7, and 11 (K4, K7 and K11) carrying acetyl groups (Hu et al., 2013, Ishibashi et al., 2009). To determine whether p300 acetylates these residues, we used mass spectrometry to map acetylation sites on H2A.Z N-terminal peptide after reaction with p300 (Figures 1F and S2A). By four minutes, 90% of all peptides were acetylated, and unacetylated peptides were not detectable in the subsequent time points. Mono-acetylated (mono-ac) peptides were only present after 4, 8, or 16 min, and the proportion decreased by half over the time course (63.6%, 36.9%, and 17.8%, respectively). In contrast, tetra-acetylated peptides were detected (14.4% and 21.1%) at the longest time points (32 and 64 min). Tandem MS analysis showed that all lysines on the N-terminus of H2A.Z (K4, K7, K11, K13, and K15) could be acetylated by p300, but K4 and K7 were preferred (Figure S2B).

In summary, we provide biochemical evidence that the lysine acetyltransferase Tip60 alone is not sufficient to acetylate H2A.Z. In contrast, we show that p300 on its own rapidly acetylates both H2A.Z peptides and H2A.Z-containing nucleosomes at multiple N-terminal lysines.

Inhibition of p300 and Tip60 Decreases H2A.Zac

To determine whether p300 and Tip60 acetylate H2A.Z in cells, we used A-485, a recently developed and highly selective and potent inhibitor for p300/CBP KAT domains (Lasko et al., 2017) (Figure 2A), as well as genetic inhibition of Tip60, p300, and CBP by siRNA in two human cancer cell lines (LNCaP and HCT116) (Figures 2B and 2C). We isolated chromatin fractions from all conditions and performed Western blot analysis. To test changes in H2A.Z acetylation, we used three antibodies against different H2A.Z acetylation forms: H2A.Ztri-ac (K4, K7 and K11), H2A.ZK4ac, and H2A.ZK7ac. We characterized the antibody substrate affinity by peptide microarray, as previously described (Rothbart et al., 2012a). We found that H2A.Ztri-ac specifically recognizes the tri-acetylated form of H2A.Z; H2A.ZK4ac binds to substrates that contain H2A.ZK4ac but does not distinguish between degrees of acetylation, whereas H2A.ZK7ac binds exclusively to mono-acetylated H2A.Z at K7 (Figure S3).

Figure 2.

Figure 2

Open in a new tab

Inhibition of Tip60 and p300 Decrease H2A.Z Acetylation in Cancer Cell Lines

(A) Western blot analysis of chromatin purified extracts from LNCaP and HCT116 cells treated with A-485 for 24 h at increasing concentrations (0.08, 0.4, 2, and 10 μM). The compound A-486, an inactive analog of A-485, was also used as negative control. The figure shows a representative image of two biological replicates (top) and densitometry quantification (bottom). Densitometry quantification of histone acetylation abundance is shown at the bottom, and it is represented as the relative amount to DMSO control in logarithmic scale. H2A.Z acetylation forms were normalized to total H2A.Z; H3K27ac and H4ac were normalized to H4. Data are represented as mean and SD (n = 2). Student's t-test was performed to compare each condition to DMSO. *p value<0.05, **p value<0.01, ***p value<0.001.

(B) Quantification of the knockdown (KD) levels of the siRNA experiments by mRNA expression (left panel) and protein abundance of TIP60 (right panel). Cells were transfected with a pool of four siRNAs for Tip60 and CBP and three siRNAs for p300 (See Table S3 for siRNA sequences). The pools were then combined accordingly for the double p300/CBP KD and triple p300/CBP/Tip60 KD. mRNA expression of Tip60, p300, and CBP was measured by real-time qPCR in cells transfected with Tip60, p300, and CBP siRNAs (left). mRNA levels of all conditions were calculated relative to untargeted siRNA control (scramble). Data are represented as mean and SD (n = 2), and Student's t-test was performed to compare each condition to scramble. Densitometry quantification of each Tip60 band was normalized to the loading control H4 and relative to scramble, and it is represented as logarithmic scale. The mean relative abundance of each band and SD was plotted and Student's t-test was performed to compare each condition to scramble. *p value<0.05, **p value<0.01, ***p value<0.001. Find list of siRNA and primer sequences in Tables S3 and S4, respectively.

(C) Western blot analysis of the chromatin fractions from LNCaP cells transfected with the scramble control (Scrble) Tip60 siRNA (siTIP60), p300, and CBP siRNAs (sip300/CBP) or p300, CBP, and Tip60 siRNAs (sip300/CBP/TIP60). The figure shows a representative image of at least two biological replicates (top) and densitometry quantification (bottom). Histone acetylation abundance was measured by densitometry and calculated relative to the scramble control. Data are represented as mean and SD (n = 2 or more). The graph is shown in a logarithmic scale, and Student's t-test was performed to compare each condition to scramble. *p value<0.05, **p value<0.01, ***p value<0.001.

(D) Weinert et al.’s acetylome mass spectrometry data after CBP/p300 perturbations, including double knockout of p300 and CBP genes (KO, dark blue) and chemical inhibition with A-485 (A-485, yellow) and compound-R (Cmpd-R, light blue) in mouse embryonic fibroblasts cells. Kasumi-1 cells, a human acute myeloid leukemia cell line, were treated with compound-R (red). Log2 SILAC normalized ratios for each detectable acetylation site of H2A.Z-1 and H2A.Z-2 isoforms were plotted. SILAC normalized ratios were calculated as explained in Weinert et al. (2018). The peptides containing K4ac are identical between isoforms, and therefore, they were analyzed as one. Error bars represent standard error of three independent replicates when available.

See also Figure S3 and Tables S1, S3, and S4.

Using these antibodies, we found that cells treated with A-485 had a strong and consistent dose-dependent reduction of the positive control H3K27ac between cell lines, whereas the less specific substrate, H4tetraAc, did not show a clear reduction (Figure 2A). Signal from the H2A.Ztri-ac antibody did not show a significant reduction in either of the cell lines, whereas H2A.ZK4ac and H2A.ZK7ac showed a clear dose-dependent decrease. At 10 μM concentration of A-485, the mono-acetylated H2A.Z antibodies showed an average reduction of 67% and 92% in LNCaP and HCT116 cells, respectively. No clear differences were detected between the reduction of H2A.ZK4ac and H2A.ZK7ac. These data suggest that inhibition of p300/CBP by A-485 preferentially reduces H2A.ZK4ac and H2A.ZK7ac mono-acetylated forms.

There are currently no selective Tip60 inhibitors commercially available. Therefore, we performed siRNA knockdown (KD) experiments to investigate the effects on H2A.Zac (Figures 2B and 2C). Tip60 mRNA levels were reduced by 87% and protein abundance by 80% following Tip60 KD (Figures 2B and 2C). H2A.Zac was significantly reduced after Tip60 KD, 58% for the triAc, 45% for K4ac, and 79% for K7ac (Figure 2C), as previously reported in Tip60 conditional knockout experiments (Li et al., 2019). We found that the tetra-acetylated form of H4 was reduced by 64%, in line with our cell-free assays. We also performed siRNA experiments for the double p300/CBP KD and a triple p300/CBP/Tip60 KD. Even though the mRNA levels of p300 and CBP in the double KD were moderately decreased (∼50% and 32%, respectively, Figure 2B), the positive control H3K27ac was reduced by 92% (Figure 2C), suggesting the HAT inhibition was enough to detect changes in the histone acetylation profile. Interestingly, Tip60 mRNA levels increased by two-fold after p300/CBP KD but this did not translate to a significant change of Tip60 protein level (Figure 2B). The double p300/CBP KD reduced all H2A.Zac forms to a similar ratio as the Tip60 KD (Figure 2C). However, the triple KD of CBP, p300 and Tip60 showed a greater reduction of H2A.Z acetylation in comparison to the single Tip60 or p300/CBP KDs (Figures 2B and 2C). Interestingly, K7 acetylation was the most reduced in all KD combinations, with 97% reduction in the triple KD.

A recent resource paper mapped the p300 acetylome in mammals using quantitative mass spectrometry after genetic or chemical modulation of p300 activity (Weinert et al., 2018). We analyzed this H2A.Z acetylation data and confirmed our in vitro and in-cell results (Figure 2D). Consistently, H2A.Z acetylation at lysines K4, K7, K11, and K13 was reduced after chemical inhibition of p300 KAT activity in both MEFs and Kasumi-1 cell lines (Figure 2D). The greatest effect was observed in the p300/CBP double knockout. Taken together, these data strongly support the conclusion that p300/CBP is a major KAT for H2A.Z in cells. We found that although Tip60 can modulate acetylation levels of H2A.Zac, it is not the sole KAT for H2A.Z and that p300/CBP is also essential for the acetylation of H2A.Z (Figure 6A).

Figure 6.

Figure 6

Open in a new tab

Model Figure of H2A.Z Acetylation Writing Mechanism

(A) Proposed models for Tip60- and p300-mediated acetylation of H2A.Z. (1) Sequential cascade: Tip60 acetylates H4, which drives recruitment of p300 and acetylation of H2A.Z. (2) Independent mechanisms: Tip60 directly targets acetylation of H2A.Z potentially during histone exchange and p300 acetylates H2A.Z at the chromatin to maintain nucleosomal acetylation.

(B) The combinatorial subgroups of H2A.Zac and H4ac at enhancers are snapshot of a dynamic mechanism for enhancer activation through three states: poised, poised-active, and active.

Interaction of p300 BD with H4ac Promotes p300-mediated H2A.Z Acetylation In Vitro

Previous studies have shown that p300 BD binds H4ac, thereby acting as a reader of histone acetylation (Delvecchio et al., 2013, Nguyen et al., 2014). Interestingly, there is high sequence homology between H4 and H2A.Z at their N-termini (Figure S4A), where the spacing of the lysines follow the same KAc(X)2–3KAc pattern, in which X represents any amino acid, which is reported to have the strongest p300 BD binding (Delvecchio et al., 2013). We questioned whether the p300 BD was also capable of reading H2A.Zac. Using histone peptide arrays printed with approximately 260 uniquely modified histone peptides (Cornett et al., 2016), including major single and combinatorial acetylations on all four core histones and the variant H2A.Z (see Table S1), we revealed that p300 BD binds exclusively to H4-acetylated peptides (Figures 3A and S4B). No binding was measured for acetylated H2A.Z or any other acetylated histone peptide in the library.

Figure 3.

Figure 3

Open in a new tab

Bromodomain Binding to H4-Acetylated Tail Enhances H2A.Z Acetylation

(A) Heatmap showing p300 BD binding preference for H2A.Z and H4 peptides from histone peptide microarray analysis. Scalebar shows the color key to the relative intensity, normalized to the peptide with the highest raw signal (H4K5acK8acK12acK16ac). Data are represented as mean normalized signal (n = 6). Array 1 was used for this experiment. The complete list of peptides included in the histone peptide microarray is in Table S1.

(B) H3 fluorography (top) and Coomassie staining (bottom) of H2A canonical nucleosomes (canonical nuc) or H4K5acK8acK12acK16ac pre-marked canonical nucleosomes (H4ac-canonical nuc) incubated with p300 over time (0, 2, 4, 8, and 16 min). Representative image of two experiments. The right plot shows the quantification of the acetylated band density corresponding to H2A/H2B/H3 in the canonical nucleosome and H4ac-canonical nucleosome over time.

(C) H3 fluorography (top) and Coomassie staining (bottom) of H2A.Z-1 nucleosomes incubated with p300 over time (0, 2, 4, 8, 16, 32, and 64 min). The right plot shows the density quantificatification of H2B/H3, H2A.Z, and H4 acetylated bands over time.

(D) Heatmap showing p300 BD binding preference for H2A.Z and H4 peptides in the presence or absence of CBP30 inhibitor from histone peptide microarray analysis. DMSO was used as vehicle control. Scalebar shows the color key to the relative intensity, normalized to the IgG positive control of each array. Data are represented as mean normalized signal (n = 6). Array 1 was used for this experiment. The complete list of peptides included in the histone peptide microarray is in Table S1.

(E) H3 fluorography (top) and Coomassie staining (bottom) of canonical, H2A.Z-1 and H4K5acK8acK12acK16ac modified canonical nucleosomes incubated with p300 in the presence of CBP30 or DMSO as indicated.

(F) Western blot analysis of HCT116 cells treated with CBP30 for 24 h at increasing concentrations (1, 2.5, 5, and 10 μM). Representative image of at least two biological replicates (top) and densitometry quantification represented in logarithmic scale (bottom). Protein abundance was calculated relative to DMSO control, and data are represented as mean and SD (n = 2). Student's t-test was performed to compare each condition to DMSO. *p value<0.05, **p value<0.01, ***p value<0.001.

See also Figure S4 and Table S1.

A feedforward mechanism of p300-dependent H3K27 acetylation through BD recruitment to H4ac has been previously proposed (Nguyen et al., 2014). Indeed, in our in vitro KAT assays, the rate of p300 acetylation on nucleosomes pre-installed with H4ac was faster than unmodified nucleosomes (Figure 3B). Thus, we hypothesized that the p300 BD interaction with H4ac may also enhance H2A.Z acetylation. To test this hypothesis, we first performed a time course radioactive KAT assay with recombinant p300 and H2A.Z-containing nucleosome substrates (Figure 3C). As expected, we observed time-dependent acetylation on all nucleosomal histones. Notably, H2A.Z acetylation trailed H4 acetylation in this time course.

We next used the p300 BD inhibitor, CBP30 (Conery et al., 2016, Hammitzsch et al., 2015), to further question whether p300 BD activity impacts H2A.Z acetylation. Using the same peptide array approach, we validated that CBP30 completely blocks the p300 BD from binding to H4-acetylated peptides (Figures 3D, S4C, and S4D). We performed in vitro KAT assays in the presence and absence of CBP30 using recombinant canonical, H2A.Z-containing, and H4ac-containing nucleosomes. Inhibition of the p300 BD resulted in decreased acetylation of all histones, including histone H2A.Z (Figure 3E). To further validate that BD recruitment enhances p300-mediated H2A.Z acetylation, we treated HCT116 cells with CBP30 for 24 h using a concentration range (1–10uM), recommended by Conery et al., 2016 (Figure 3F). We observed a reduction in H3K27ac levels, as previously reported (Raisner et al., 2018). Notably, there was also a decrease in H2A.Zac levels, where H2A.ZK7ac showed a striking reduction of up to 90%. Taken together, these data suggest that acetylation of H2A.Z by p300 is enhanced upon recruitment to H4-acetylated nucleosomes via the p300 BD.

H2A.Zac and H4K5ac Co-occur at Promoter Regions

We next sought to determine whether H2A.Zac- and H4ac-marked nucleosomes co-exist in the chromatin. To address this, we determined the genomic distribution of both marks using publicly available ChIP-seq data for H2A.Zac and H4K5ac in LNCaP cells (Table S2). The specificity of both antibodies was characterized with histone peptide arrays (Rothbart et al., 2012a, Dickson et al., 2016) (Figure S3A for H2A.Ztri-ac and http://www.histoneantibodies.com/ for H4K5ac). As previously reported (Rothbart et al., 2012b, Rothbart et al., 2015), the H4K5ac antibody bound preferentially to H4 peptides that also have acetylation at other sites forming di-, tri-, and tetra-acetylation states. Thus, we considered H4K5ac ChIP-seq data as reporting on H4 N-terminal tail poly-acetylation (referred to herein as H4ac), a signature consistent with the binding activity of p300 BD (Figure 3A).

We first investigated the co-occurrence of H2A.Zac and H4ac ChIP-seq peaks genome-wide and found a significant overlap, with 51.8% of H2A.Zac peaks coinciding with H4ac (Figure 4A). To consider the predicted functional properties, we used ChromHMM (Ernst and Kellis, 2012) to demarcate six different genomic regulatory regions based on the information from six chromatin marks (Figure S5A and Table S2). We found that H4ac had a similar functional distribution pattern as H2A.Zac (Valdes-Mora et al., 2017), where it was significantly enriched at promoters and enhancers and depleted at polycomb and transcribed regions (Figure S5B). To address whether the epigenetic context impacts the coexistence of H2A.Zac and H4ac, we also performed ChromHMM analysis of the overlapping marked peaks (23,904 regions, called common peaks) and unique marked peaks for H2A.Zac or H4ac (20,876 and 15,460 regions, called H2A.Zac- or H4ac-unique peaks, respectively) (Figure 4B). We found that the common peaks were most significantly located at active promoters: 51.64% of the common peaks (Figure 4B, upper panel) and 68.74% of all active promoters (Figure 4C). In contrast, the uniquely marked regions were more prevalent at enhancers (Figure 4B, middle and bottom panels), where most of the H2A.Zac-unique peaks occurred at active enhancer regions (Figure 4B, middle panel), whereas H4ac-unique peaks were mostly enriched at poised enhancers (Figure 4B, bottom panel). However, at distal regulatory regions, we observed that both common and unique regions were present in different proportions (Figure 4D), suggesting a dynamic interplay of these two marks.

Figure 4.

Figure 4

Open in a new tab

Crosstalk and Genomic Localization of H2A.Zac and H4K5ac

(A) Genomic overlap of all H2A.Zac and H4ac ChIP-seq peaks. Minimum overlap equals 1bp (left panel). Observed vs expected logarithmic fold enrichment graphs using Genomic Association Test (GAT) of H2AZ.ac over H4ac and reversely (right panel). *p value< 0.0001.

(B) GAT of H2AZ.ac and H4ac ChIP-seq peaks and ChromHMM regions in LNCaP cells. The three groups examined are H2A.Zac and H4K5ac common peaks, H2A.Zac-unique peaks, and H4K5ac-unique peaks. Pie charts representing the percentage of marked peaks falling in each ChromHMM state (left panel). Observed vs expected log-fold enrichment graphs (right panel). *p < 0.0001.

(C and D) Heatmaps showing H2A.Zac and H4ac presence across active promoters (n = 12,770) (C) and active enhancer (n = 27,898) and poised enhancers (n = 17,354) (D). Heatmaps are divided into the same three groups than panel B and order based on H2A.Zac intensity from top to bottom. ChIP-seq signal is centered at the TSS in active promoters and at the DNAseI midpoint in enhancers. The scale bars show the color key of the ChIP-seq intensity (counts per million mapped reads, CPM). The same scale is used for each ChIP-seq across all regulatory regions.

(E) Heatmaps showing H2A.Z, p300, and H3K27ac enrichment across active enhancers (left) and poised enhancers (right). Heatmaps are divided, ordered, and centered similarly to panel (D).

(F) Nucleosome occupancy and methylation sequencing (NOMe-seq) plots representing the average DNA methylation (0–1) and chromatin accessibility (1 minus GpC methylation ratio) ratios across active (left hand side) and poised enhancers (right hand side) divided into the same three groups than panel (B). The average of all active and poised enhancers is also included.

See also Figure S5 and Table S2.

Epigenetic Characteristics of Enhancers with Differential Distribution of H2A.Zac and H4ac

In order to further define the patterns of H2A.Zac- and H4ac-marked nucleosomes at enhancers, we examined different epigenetic features at these regions, including H2A.Z, p300, and H3K27ac, DNA methylation, and chromatin accessibility (Table S2). ChIP-seq data was split by H2A.Zac and H4ac signatures and ordered by H2A.Zac intensity at both poised and active enhancers (Figure 4E). Notably, we found that enhancers lacking H2A.Zac were not enriched for total H2A.Z, suggesting that enhancers with an H4ac-unique signature are also void of H2A.Z. Second, in agreement with a p300 BD-mediated recruitment mechanism proposed by this study and others (Conery et al., 2016, Manning et al., 2001), p300 showed differential occupancy between groups at both active and poised enhancers; in particular, the group lacking H4ac showed less p300 occupancy. Finally, H3K27ac, a histone PTM signature characteristic of active enhancers, did not show differential distribution between the groups, although H3K27ac intensity correlated with H2A.Zac signal. These data suggest a common pattern of acetylation of H3K27ac and H2A.Zac that may be established by p300.

We next used Nucleosome Occupancy and Methylation sequencing (NOMe-seq) data to study DNA methylation and chromatin accessibility at these enhancer sites (Kelly et al., 2012). We found that H2A.Zac-unique enhancers had less DNA methylation and were more accessible than the average of all active and poised enhancers. In contrast, H4ac-unique active and poised enhancers had more methylation and were less accessible (Figure 4F). Enhancers carrying both H2A.Zac and H4ac had similar levels of DNA methylation and accessibility as the average enhancers. In summary, we found that enhancers with differential H2A.Zac and H4ac signatures are associated with distinct epigenetic features (Figure 6B).

Acetylation Dynamics between H2A.Z and H4 at Enhancers Is Associated with Transcription Elongation

Chromatin accessibility and DNA methylation are indicative of enhancer activity (Valdes-Mora et al., 2017). To characterize the functional status of each enhancer H4/H2A.Z acetylation signature, we examined RNA polymerase II (RNApolII) recruitment and enhancer RNA (eRNA) production (Kim et al., 2010) as additional markers of enhancer function (Table S2). Figure 5A shows RNApolII ChIP-seq signal and Global Run-on sequencing (GRO-seq) data across active and poised enhancers. eRNA production is measured as the bidirectional RNA expression detected by GRO-seq around the DNAseI midpoint. Despite no major differences between H2A.Zac/H4ac signatures in eRNA production and the presence of RNAPolII flanking DNAseI sites, we did reveal evidence of differential transcription across the entire 4kb region plotted (Figure 5A). H2A.Zac-unique enhancers had less regional transcriptional activity than the other two groups (common and H4ac-unique) in both active and poised enhancers.

Figure 5.

Figure 5

Open in a new tab

Transcription Elongation Influences H2A.Zac and H4K5ac Landscape at Enhancers

(A) Heatmaps showing phosphoserine 5 RNA polymerase II (RNAPolII) ChIP-seq and global run-on sequencing (GRO-seq) signal across active and poised enhancers. The three groups examined are H2A.Zac and H4ac overlapping peaks (Common, yellow), H2A.Zac-unique peaks (H2A.Zac-unique, light blue), and H4ac-unique peaks (H4ac-unique, dark blue) at active (left) and poised (right) enhancers that are at least 2kb away from any TSS. Heatmaps are ordered based on H2A.Zac intensity from top to bottom. The scale bars show the color key of the ChIP-seq and GRO-seq intensities (counts per million mapped reads, CPM).

(B) Bar plots showing the proportion of intragenic (black) and intergenic (gray) active and poised enhancers in each H2A.Zac/H4ac group. The red dashed line marks the proportion of intergenic and intragenic enhancers found in all active and poised enhancers.

(C) Box plots comparing gene expression as logarithmic transcripts per million (logTPM) measured by RNA-seq of the genes contained on each intragenic active (left) or poised enhancers for each H2A.Zac/H4ac group and all the genes containing at least one enhancer (gray box plot).

(D) Bidirectional enhancer RNAs (eRNAs) profile plots (plus, red, and negative, blue-strands) of GROseq data around intragenic and intergenic active enhancers (top) and poised enhancers (bottom) of the H2A.Zac/H4ac groups. The plots show one representative replicate out of four. Profiles are centered at the DNAse I midpoint.

See also Figure S5.

To confirm that the observed transcription signal at expanded enhancers was due to transcription elongation, we grouped the enhancers according to their location inside or outside genes and compared their proportion with the rest of the enhancers (Figure 5B). A higher proportion of H4ac-unique enhancers were located at intragenic regions. H2A.Zac-unique active enhancers did not show differential localisation, whereas H2A.Zac-unique poised enhancers showed a higher presence at intergenic regions (Figure 5B). Common enhancers had similar inter/intragenic distribution to all detectable active and poised enhancers. We also found that genes with H2A.Zac-unique enhancers had significantly lower transcription compared with all genes containing either active or poised enhancers, whereas H4ac-unique enhancers were mostly present at highly expressed genes (Figure 5C). Moreover, the nascent RNA profiles measured by GROseq at intragenic enhancers validated the gene expression pattern (Figures 5D and S5C).

Taken together, these results demonstrate a particular H2A.Zac and H4ac pattern, or enhancer code, that correlates with their genomic localization (inter or intragenic), regional transcriptional activity, chromatin features (p300, chromatin accessibility), and DNA methylation (Figure 6B).

Discussion

A key step to understanding histone PTM function is the characterization of the enzymes and effector proteins that write, erase, and read these marks. Although much progress has been made identifying the core histone PTM modifiers (Voss and Thomas, 2018), the specific enzymes that target histone variants PTMs have not been well studied. In particular, the KATs responsible for acetylation of H2A.Z in mammals are still unresolved. Here, we report that in addition to Tip60, p300 is able to acetylate the histone variant H2A.Z and that p300-dependent H2A.Z acetylation is enhanced through BD recognition of H4ac.

Unlike Tip60, p300 has not been described to interact with H2A.Z (Choi et al., 2009, Link et al., 2018, Obri et al., 2014, Vardabasso et al., 2015), as it potentially follows a hit-and-run kinetic mechanism (Liu et al., 2008). Tip60, however, has been described as the KAT candidate responsible for acetylation of H2A.Z (Corujo and Buschbeck, 2018, Giaimo et al., 2019, Sevilla and Binda, 2014). Tip60 is part of a larger complex, involved in H2A.Z histone exchange (Altaf et al., 2010, Cai et al., 2003, Keogh et al., 2006, Kusch et al., 2004), which makes it difficult to assess whether Tip60 alone is able to acetylate H2A.Z. In fact, our in vitro results show that Tip60 acetylates peptide and nucleosomal H4 but does not efficiently acetylate H2A.Z. However, we and others (Dalvai et al., 2012, Li et al., 2019) found that genetic inhibition of Tip60 decreases H2A.Zac levels, suggesting that Tip60, in its cellular context, facilitates H2A.Z acetylation. Of note, complete knockout of Tip60 does not abolish H2A.Zac (Li et al., 2019), and other reports found that acetylation of H2A.Z was independent of Tip60 at some locus-specific promoter (Bellucci et al., 2013, Semer et al., 2019), supporting our finding that p300/CBP is also required for the acetylation of H2A.Z. Based on our new data, we propose a model whereby acetylation of H2A.Z is the result of a sequential cascade initiated by Tip60-mediated acetylation of H4, leading to the recruitment of p300 via its BD to acetylate H2A.Z. Alternatively, Tip60 and p300/CBP may acetylate H2A.Z directly through independent pathways (Figure 6A).

We provide biochemical evidence that the presence of acetylated H4 in the nucleosome can have impact on p300-mediated acetylation of H2A.Z, suggesting that these two marks are co-localized. It was previously shown by immunoprecipitation that H2A.Z nucleosomes are enriched for H4ac (Draker et al., 2012, Myers et al., 2006). Based on our analysis of ChIP-seq data, we found that H2A.Zac and H4ac co-occurred predominantly at promoters and enhancers. However, at a third of all enhancers, these marks did not co-occur. After dissecting the epigenetic features of each sub-group of enhancers, we propose a dynamic mechanism for enhancer activation through three states of activity (Figure 6B): poised, poised-active, and active. Each state has a preferential H4ac/H2A.Zac combination that would initiate from H4ac-marked nucleosomes at poised enhancers that enables the recruitment of p300 through its BD. This may trigger H2A.Z acetylation through p300 in a poised/active state and deacetylation of H4 in a fully enhancer-activated state.

Additionally, we also find different transcriptional features of each state. H2A.Zac-unique enhancers are enriched at intergenic regions or within lowly expressed genes; conversely, H4ac-unique enhancers are present in intragenic regions and are associated with highly transcribed genes, whereas common enhancers are characterized by an intermediate state regarding transcription elongation. The lack of H2A.Zac at intragenic enhancers of highly expressed genes could be due to the previously reported active removal of H2A.Z at gene bodies during transcription elongation (Hardy et al., 2009, Lashgari et al., 2017). It is therefore interesting to note that H2A.Zac-marked active enhancers are not present within the gene they activate, as transcription elongation of the gene would remove H2A.Z from the enhancer, suggesting that H2A.Zac enhancers are likely acting as trans-regulatory elements. In this regard, a recent study showed that functionally active intragenic enhancers can attenuate transcription of the host gene (Cinghu et al., 2017), supporting our observation.

In contrast, the lack of H4ac at H2A.Zac-unique enhancers is less clear. Genome-wide mapping of KATs and lysine deacetylases (KDACs) have shown that both enzyme families are targeted to active genes and correlate with transcription levels (Wang et al., 2009), which suggests a high turnover rate of histone acetylation at highly transcribed genes compared with lowly transcribed genes or intergenic regions. Weinert et al. data reported a higher deacetylation rate of H4 compared with H2A.Z (Weinert et al., 2018). We, therefore, propose that these different kinetics could partially explain the lack of H4ac at these H2A.Zac regions. However, we cannot rule out that p300 and Tip60 are able to acetylate H2A.Z without the presence of H4ac at these particular sites.

Collectively, we demonstrate that p300/CBP, complementary to Tip60, are essential KATs for the acetylation of H2A.Z, answering the long-standing question of what enzymes are responsible for this important modification. We reveal a mechanism of histone PTM crosstalk and define an enhancer code based on the patterning of H4ac and H2A.Zac. Both p300 and H2A.Z have been implicated in many human cancers (reviewed in Iyer et al., 2004, Vardabasso et al., 2014); therefore, this study provides p300 as an additional target for H2A.Zac-based chromatin therapy in cancer.

Limitations of the Study

In this study we demonstrate that p300-BD interaction with H4ac enhances H2A.Z acetylation using an inhibitor of p300 BD. However, we were not able to directly compare p300-mediated acetylation in a nucleosomal context with pre-installed H4ac/H2A.Z nucleosomes due to the technical difficulty in generating recombinant H4ac/H2A.Z combinatorial nucleosomes in vitro. Additionally, we found that H2A.Zac levels are reduced after inhibition of both Tip60 and p300, but we were unable in this study to determine if the mechanism that results in H2A.Zac involves a sequential cascade through H4ac or occurs through independent pathways. Future studies will focus on resolving the interplay and order of events that are associated with Tip60 and p300 in driving the acetylation of H2A.Z.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.

Acknowledgments

We thank Dr. Ozren Bogdanovic and Dr. Paul Timpson for their review and feedback on the manuscript. This work was funded by National Health and Medical Research Council, Australia (NHMRC) project grant (#1144574) (S.J.C and F.V.M); National Institutes of Health, USA (NIH) project grant (R35GM124736) (S.B.R); NHMRC Fellowship (#1063559) (S.J.C.); Cancer Institute NSW Career Development fellowship (CDF181218, from 2019) (F.V.M); and UNSW Sydney University Tuition Fee Scholarship (TFS), Australia (Y.C.S.). We thank Epicypher, Inc. for gifting nucleosome reagents for this study.

Author Contributions

Conception and design: Y.C.S., F.V.M., S.J.C., E.M.C., and S.B.R.; Biochemical experiments: E.M.C., Y.C.S., J.H., E.C.S., and R.M.V.; Mass-spec experiments: K.K. and E.M.C.; In-cell experiments: Y.C.S., D.M., and G.C.S; NGS analysis: Y.C.S.; Interpretation of the data: Y.C.S., F.V.M., S.J.C., E.M.C., and S.B.R.; Writing and review of manuscript Y.C.S., F.V.M., S.J.C., E.M.C., R.M.V., and S.B.R.. All authors have read and approved the final manuscript.

Declaration of Interests

S.B.R. has served in a compensated consulting role for EpiCypher, Inc. All other authors declare no competing interest.

Published: November 22, 2019

Footnotes

Supplemental Information can be found online at https://doi.org/10.1016/j.isci.2019.10.053.

Contributor Information

Scott B. Rothbart, Email: scott.rothbart@vai.org.

Susan J. Clark, Email: s.clark@garvan.org.au.

Fátima Valdés-Mora, Email: f.valdesmora@garvan.org.au.

Supplemental Information

Document S1. Transparent Methods, Figures S1–S5, and Tables S2–S4
mmc1.pdf (3.6MB, pdf)
Table S1. List of Peptides in Array 1 and 2, Related to Figures 3 and S3
mmc2.xlsx (28.5KB, xlsx)

References

  1. Altaf M., Auger A., Monnet-Saksouk J., Brodeur J., Piquet S., Cramet M., Bouchard N., Lacoste N., Utley R.T., Gaudreau L. NuA4-dependent acetylation of nucleosomal histones H4 and H2A directly stimulates incorporation of H2A.Z by the SWR1 complex. J. Biol. Chem. 2010;285:15966–15977. doi: 10.1074/jbc.M110.117069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bellucci L., Dalvai M., Kocanova S., Moutahir F., Bystricky K. Activation of p21 by HDAC inhibitors requires acetylation of H2A.Z. PLoS One. 2013;8:e54102. doi: 10.1371/journal.pone.0054102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bruce, Myers, Mantouvalou, Lefevre, Greaves, Bonifer, Tremethick, Thorne, Crane-Robinson The replacement histone H2A.Z in a hyperacetylated form is a feature of active genes in the chicken. Nucleic Acids Res. 2005;33:5633–5639. doi: 10.1093/nar/gki874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brunelle, Nordell Markovits, Rodrigue, Lupien, Jacques, Gevry The histone variant H2A.Z is an important regulator of enhancer activity. Nucleic Acids Res. 2015;43:9742–9756. doi: 10.1093/nar/gkv825. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cai Y., Jin J., Tomomori-Sato C., Sato S., Sorokina I., Parmely T.J., Conaway R.C., Conaway J.W. Identification of new subunits of the multiprotein mammalian TRRAP/TIP60-containing histone acetyltransferase complex. J. Biol. Chem. 2003;278:42733–42736. doi: 10.1074/jbc.C300389200. [DOI] [PubMed] [Google Scholar]
  6. Choi J., Heo K., An W. Cooperative action of TIP48 and TIP49 in H2A.Z exchange catalyzed by acetylation of nucleosomal H2A. Nucleic Acids Res. 2009;37:5993–6007. doi: 10.1093/nar/gkp660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cinghu S., Yang P., Kosak J.P., Conway A.E., Kumar D., Oldfield A.J., Adelman K., Jothi R. Intragenic enhancers attenuate host gene expression. Mol. Cell. 2017;68:104–117.e6. doi: 10.1016/j.molcel.2017.09.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Colino-Sanguino Y., Clark S.J., Valdes-Mora F. H2A.Z acetylation and transcription: ready, steady, go! Epigenomics. 2016;8:583–586. doi: 10.2217/epi-2016-0016. [DOI] [PubMed] [Google Scholar]
  9. Conery A.R., Centore R.C., Neiss A., Keller P.J., Joshi S., Spillane K.L., Sandy P., Hatton C., Pardo E., Zawadzke L. Bromodomain inhibition of the transcriptional coactivators CBP/EP300 as a therapeutic strategy to target the IRF4 network in multiple myeloma. Elife. 2016;5:e10483. doi: 10.7554/eLife.10483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cornett E.M., Dickson B.M., Vaughan R.M., Krishnan S., Trievel R.C., Strahl B.D., Rothbart S.B. Substrate specificity profiling of histone-modifying enzymes by peptide microarray. Methods Enzymol. 2016;574:31–52. doi: 10.1016/bs.mie.2016.01.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Corujo D., Buschbeck M. Post-Translational modifications of H2A histone variants and their role in cancer. Cancers (Basel) 2018;10:59. doi: 10.3390/cancers10030059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Creyghton M.P., Cheng A.W., Welstead G.G., Kooistra T., Carey B.W., Steine E.J., Hanna J., Lodato M.A., Frampton G.M., Sharp P.A. Histone H3K27ac separates active from poised enhancers and predicts developmental state. Proc. Natl. Acad. Sci. U S A. 2010;107:21931–21936. doi: 10.1073/pnas.1016071107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dalvai M., Bellucci L., Fleury L., Lavigne A.C., Moutahir F., Bystricky K. H2A.Z-dependent crosstalk between enhancer and promoter regulates Cyclin D1 expression. Oncogene. 2012;32:4243–4251. doi: 10.1038/onc.2012.442. [DOI] [PubMed] [Google Scholar]
  14. Dalvai M., Fleury L., Bellucci L., Kocanova S., Bystricky K. TIP48/Reptin and H2A.Z requirement for initiating chromatin remodeling in estrogen-activated transcription. PLoS Genet. 2013;9:e1003387. doi: 10.1371/journal.pgen.1003387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dancy B.M., Cole P.A. Protein lysine acetylation by p300/CBP. Chem. Rev. 2015;115:2419–2452. doi: 10.1021/cr500452k. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Delvecchio M., Gaucher J., Aguilar-Gurrieri C., Ortega E., Panne D. Structure of the p300 catalytic core and implications for chromatin targeting and HAT regulation. Nat. Struct. Mol. Biol. 2013;20:1040–1046. doi: 10.1038/nsmb.2642. [DOI] [PubMed] [Google Scholar]
  17. Dickson B.M., Cornett E.M., Ramjan Z., Rothbart S.B. ArrayNinja: an open source platform for unified planning and analysis of microarray experiments. Methods Enzymol. 2016;574:53–77. doi: 10.1016/bs.mie.2016.02.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Doyon Y., Selleck W., Lane W.S., Tan S., Cote J. Structural and functional conservation of the NuA4 histone acetyltransferase complex from yeast to humans. Mol. Cell Biol. 2004;24:1884–1896. doi: 10.1128/MCB.24.5.1884-1896.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Draker R., Ng M.K., Sarcinella E., Ignatchenko V., Kislinger T., Cheung P. A combination of H2A.Z and H4 acetylation recruits Brd2 to chromatin during transcriptional activation. PLoS Genet. 2012;8:e1003047. doi: 10.1371/journal.pgen.1003047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Dryhurst D., Ishibashi T., Rose K.L., Eirin-Lopez J.M., McDonald D., Silva-Moreno B., Veldhoen N., Helbing C.C., Hendzel M.J., Shabanowitz J. Characterization of the histone H2A.Z-1 and H2A.Z-2 isoforms in vertebrates. BMC Biol. 2009;7:86. doi: 10.1186/1741-7007-7-86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Dryhurst, McMullen, Fazli, Rennie, Ausio Histone H2A.Z prepares the prostate specific antigen (PSA) gene for androgen receptor-mediated transcription and is upregulated in a model of prostate cancer progression. Cancer Lett. 2012;315:38–47. doi: 10.1016/j.canlet.2011.10.003. [DOI] [PubMed] [Google Scholar]
  22. Ernst, Kellis ChromHMM: automating chromatin-state discovery and characterization. Nat Methods. 2012;9:215–216. doi: 10.1038/nmeth.1906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Giaimo B.D., Ferrante F., Herchenrother A., Hake S.B., Borggrefe T. The histone variant H2A.Z in gene regulation. Epigenetics Chromatin. 2019;12:37. doi: 10.1186/s13072-019-0274-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Giaimo B.D., Ferrante F., Vallejo D.M., Hein K., Gutierrez-Perez I., Nist A., Stiewe T., Mittler G., Herold S., Zimmermann T. Histone variant H2A.Z deposition and acetylation directs the canonical Notch signaling response. Nucleic Acids Res. 2018;46:8197–8215. doi: 10.1093/nar/gky551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Halley, Kaplan, Wang, Kobor, Rine Roles for H2A.Z and Its Acetylation in GAL1 Transcription and Gene Induction, but Not GAL1-Transcriptional Memory. PLoS Biol. 2010;8:e1000401. doi: 10.1371/journal.pbio.1000401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Hammitzsch A., Tallant C., Fedorov O., O'Mahony A., Brennan P.E., Hay D.A., Martinez F.O., Al-Mossawi M.H., de Wit J., Vecellio M. CBP30, a selective CBP/p300 bromodomain inhibitor, suppresses human Th17 responses. Proc. Natl. Acad. Sci. U S A. 2015;112:10768–10773. doi: 10.1073/pnas.1501956112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hardy S., Jacques P.E., Gevry N., Forest A., Fortin M.E., Laflamme L., Gaudreau L., Robert F. The euchromatic and heterochromatic landscapes are shaped by antagonizing effects of transcription on H2A.Z deposition. PLoS Genet. 2009;5:e1000687. doi: 10.1371/journal.pgen.1000687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Hilton I.B., D'Ippolito A.M., Vockley C.M., Thakore P.I., Crawford G.E., Reddy T.E., Gersbach C.A. Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers. Nat. Biotechnol. 2015;33:510–517. doi: 10.1038/nbt.3199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hu G., Cui K., Northrup D., Liu C., Wang C., Tang Q., Ge K., Levens D., Crane-Robinson C., Zhao K. H2A.Z facilitates access of active and repressive complexes to chromatin in embryonic stem cell self-renewal and differentiation. Cell Stem Cell. 2013;12:180–192. doi: 10.1016/j.stem.2012.11.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ishibashi T., Dryhurst D., Rose K.L., Shabanowitz J., Hunt D.F., Ausio J. Acetylation of vertebrate H2A.Z and its effect on the structure of the nucleosome. Biochemistry. 2009;48:5007–5017. doi: 10.1021/bi900196c. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ito T., Ikehara T., Nakagawa T., Kraus W.L., Muramatsu M. p300-mediated acetylation facilitates the transfer of histone H2A-H2B dimers from nucleosomes to a histone chaperone. Genes Dev. 2000;14:1899–1907. [PMC free article] [PubMed] [Google Scholar]
  32. Ito S., Kayukawa N., Ueda T., Taniguchi H., Morioka Y., Hongo F., Ukimura O. MRGBP promotes AR-mediated transactivation of KLK3 and TMPRSS2 via acetylation of histone H2A.Z in prostate cancer cells. Biochim. Biophys. Acta Gene Regul. Mech. 2018;1861:794–802. doi: 10.1016/j.bbagrm.2018.07.014. [DOI] [PubMed] [Google Scholar]
  33. Iyer N.G., Ozdag H., Caldas C. p300/CBP and cancer. Oncogene. 2004;23:4225–4231. doi: 10.1038/sj.onc.1207118. [DOI] [PubMed] [Google Scholar]
  34. Jeong K.W., Kim K., Situ A.J., Ulmer T.S., An W., Stallcup M.R. Recognition of enhancer element-specific histone methylation by TIP60 in transcriptional activation. Nat. Struct. Mol. Biol. 2011;18:1358–1365. doi: 10.1038/nsmb.2153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Jin, Yu, Wang, Zhang, Kasper, Lee, Wang, Brindle, Dent, Ge Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in nuclear receptor transactivation. EMBO J. 2011;30:249–262. doi: 10.1038/emboj.2010.318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Kamakaka, Biggins Histone variants: deviants? Genes Dev. 2005;19:295–310. doi: 10.1101/gad.1272805. [DOI] [PubMed] [Google Scholar]
  37. Kelly T.K., Liu Y., Lay F.D., Liang G., Berman B.P., Jones P.A. Genome-wide mapping of nucleosome positioning and DNA methylation within individual DNA molecules. Genome Res. 2012;22:2497–2506. doi: 10.1101/gr.143008.112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Keogh M.C., Mennella T.A., Sawa C., Berthelet S., Krogan N.J., Wolek A., Podolny V., Carpenter L.R., Greenblatt J.F., Baetz K. The Saccharomyces cerevisiae histone H2A variant Htz1 is acetylated by NuA4. Genes Dev. 2006;20:660–665. doi: 10.1101/gad.1388106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Kim T.K., Hemberg M., Gray J.M., Costa A.M., Bear D.M., Wu J., Harmin D.A., Laptewicz M., Barbara-Haley K., Kuersten S. Widespread transcription at neuronal activity-regulated enhancers. Nature. 2010;465:182–187. doi: 10.1038/nature09033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Kimura, Horikoshi Tip60 acetylates six lysines of a specific class in core histones in vitro. Genes Cells. 1998;3:789–800. doi: 10.1046/j.1365-2443.1998.00229.x. [DOI] [PubMed] [Google Scholar]
  41. Ku, Jaffe, Koche, Rheinbay, Endoh, Koseki, Carr, Bernstein H2A.Z landscapes and dual modifications in pluripotent and multipotent stem cells underlie complex genome regulatory functions. Genome Biol. 2012;13:R85. doi: 10.1186/gb-2012-13-10-r85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Kusch T., Florens L., Macdonald W.H., Swanson S.K., Glaser R.L., Yates J.R., 3rd, Abmayr S.M., Washburn M.P., Workman J.L. Acetylation by Tip60 is required for selective histone variant exchange at DNA lesions. Science. 2004;306:2084–2087. doi: 10.1126/science.1103455. [DOI] [PubMed] [Google Scholar]
  43. Lashgari A., Millau J.F., Jacques P.E., Gaudreau L. Global inhibition of transcription causes an increase in histone H2A.Z incorporation within gene bodies. Nucleic Acids Res. 2017;45:12715–12722. doi: 10.1093/nar/gkx879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Lasko L.M., Jakob C.G., Edalji R.P., Qiu W., Montgomery D., Digiammarino E.L., Hansen T.M., Risi R.M., Frey R., Manaves V. Discovery of a selective catalytic p300/CBP inhibitor that targets lineage-specific tumours. Nature. 2017;550:128–132. doi: 10.1038/nature24028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Li M.L., Jiang Q., Bhanu N.V., Wu J., Li W., Garcia B.A., Greenberg R.A. Phosphorylation of TIP60 suppresses 53BP1 localization at DNA damage sites. Mol. Cell Biol. 2019;39 doi: 10.1128/MCB.00209-18. e00209–18. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Link S., Spitzer R.M.M., Sana M., Torrado M., Volker-Albert M.C., Keilhauer E.C., Burgold T., Punzeler S., Low J.K.K., Lindstrom I. PWWP2A binds distinct chromatin moieties and interacts with an MTA1-specific core NuRD complex. Nat. Commun. 2018;9:4300. doi: 10.1038/s41467-018-06665-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Liu X., Wang L., Zhao K., Thompson P.R., Hwang Y., Marmorstein R., Cole P.A. The structural basis of protein acetylation by the p300/CBP transcriptional coactivator. Nature. 2008;451:846–850. doi: 10.1038/nature06546. [DOI] [PubMed] [Google Scholar]
  48. Manning E.T., Ikehara T., Ito T., Kadonaga J.T., Kraus W.L. p300 forms a stable, template-committed complex with chromatin: role for the bromodomain. Mol. Cell Biol. 2001;21:3876–3887. doi: 10.1128/MCB.21.12.3876-3887.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Millar C.B., Xu F., Zhang K., Grunstein M. Acetylation of H2AZ Lys 14 is associated with genome-wide gene activity in yeast. Genes Dev. 2006;20:711–722. doi: 10.1101/gad.1395506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Myers F.A., Lefevre P., Mantouvalou E., Bruce K., Lacroix C., Bonifer C., Thorne A.W., Crane-Robinson C. Developmental activation of the lysozyme gene in chicken macrophage cells is linked to core histone acetylation at its enhancer elements. Nucleic Acids Res. 2006;34:4025–4035. doi: 10.1093/nar/gkl543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Narkaj K., Stefanelli G., Wahdan M., Azam A.B., Ramzan F., Steininger C.F.D., Jr., Walters B.J., Zovkic I.B. Blocking H2A.Z incorporation via Tip60 inhibition promotes systems consolidation of fear memory in mice. eNeuro. 2018;5:1. doi: 10.1523/ENEURO.0378-18.2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Nguyen U.T., Bittova L., Muller M.M., Fierz B., David Y., Houck-Loomis B., Feng V., Dann G.P., Muir T.W. Accelerated chromatin biochemistry using DNA-barcoded nucleosome libraries. Nat. Methods. 2014;11:834–840. doi: 10.1038/nmeth.3022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Obri A., Ouararhni K., Papin C., Diebold M.L., Padmanabhan K., Marek M., Stoll I., Roy L., Reilly P.T., Mak T.W. ANP32E is a histone chaperone that removes H2A.Z from chromatin. Nature. 2014;505:648–653. doi: 10.1038/nature12922. [DOI] [PubMed] [Google Scholar]
  54. Ogryzko V.V., Schiltz R.L., Russanova V., Howard B.H., Nakatani Y. The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell. 1996;87:953–959. doi: 10.1016/s0092-8674(00)82001-2. [DOI] [PubMed] [Google Scholar]
  55. Raisner R., Kharbanda S., Jin L., Jeng E., Chan E., Merchant M., Haverty P.M., Bainer R., Cheung T., Arnott D. Enhancer activity requires CBP/P300 bromodomain-dependent histone H3K27 acetylation. Cell Rep. 2018;24:1722–1729. doi: 10.1016/j.celrep.2018.07.041. [DOI] [PubMed] [Google Scholar]
  56. Rothbart S.B., Dickson B.M., Raab J.R., Grzybowski A.T., Krajewski K., Guo A.H., Shanle E.K., Josefowicz S.Z., Fuchs S.M., Allis C.D. An interactive database for the assessment of histone antibody specificity. Mol. Cell. 2015;59:502–511. doi: 10.1016/j.molcel.2015.06.022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Rothbart S.B., Krajewski K., Strahl B.D., Fuchs S.M. Peptide microarrays to interrogate the “histone code”. Methods Enzymol. 2012;512:107–135. doi: 10.1016/B978-0-12-391940-3.00006-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Rothbart S.B., Lin S., Britton L.M., Krajewski K., Keogh M.C., Garcia B.A., Strahl B.D. Poly-acetylated chromatin signatures are preferred epitopes for site-specific histone H4 acetyl antibodies. Sci. Rep. 2012;2:489. doi: 10.1038/srep00489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Schiltz R.L., Mizzen C.A., Vassilev A., Cook R.G., Allis C.D., Nakatani Y. Overlapping but distinct patterns of histone acetylation by the human coactivators p300 and PCAF within nucleosomal substrates. J. Biol. Chem. 1999;274:1189–1192. doi: 10.1074/jbc.274.3.1189. [DOI] [PubMed] [Google Scholar]
  60. Semer M., Bidon B., Larnicol A., Caliskan G., Catez P., Egly J.M., Coin F., Le May N. DNA repair complex licenses acetylation of H2A.Z.1 by KAT2A during transcription. Nat. Chem. Biol. 2019;15:992–1000. doi: 10.1038/s41589-019-0354-y. [DOI] [PubMed] [Google Scholar]
  61. Sevilla A., Binda O. Post-translational modifications of the histone variant H2AZ. Stem Cell Res. 2014;12:289–295. doi: 10.1016/j.scr.2013.11.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Suto R.K., Clarkson M.J., Tremethick D.J., Luger K. Crystal structure of a nucleosome core particle containing the variant histone H2A.Z. Nat. Struct. Biol. 2000;7:1121–1124. doi: 10.1038/81971. [DOI] [PubMed] [Google Scholar]
  63. Valdes-Mora F., Gould C.M., Colino-Sanguino Y., Qu W., Song J.Z., Taylor K.M., Buske F.A., Statham A.L., Nair S.S., Armstrong N.J. Acetylated histone variant H2A.Z is involved in the activation of neo-enhancers in prostate cancer. Nat. Commun. 2017;8:1346. doi: 10.1038/s41467-017-01393-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Valdes-Mora, Song, Statham, Strbenac, Robinson, Nair, Patterson, Tremethick, Stirzaker, Clark Acetylation of H2A.Z is a key epigenetic modification associated with gene deregulation and epigenetic remodeling in cancer. Genome Res. 2012;22:307–321. doi: 10.1101/gr.118919.110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Vardabasso C., Gaspar-Maia A., Hasson D., Punzeler S., Valle-Garcia D., Straub T., Keilhauer E.C., Strub T., Dong J., Panda T. Histone variant H2A.Z.2 mediates proliferation and drug sensitivity of malignant melanoma. Mol. Cell. 2015;59:75–88. doi: 10.1016/j.molcel.2015.05.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Vardabasso C., Hasson D., Ratnakumar K., Chung C.Y., Duarte L.F., Bernstein E. Histone variants: emerging players in cancer biology. Cell Mol. Life Sci. 2014;71:379–404. doi: 10.1007/s00018-013-1343-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  67. Voss A.K., Thomas T. Histone lysine and genomic targets of histone acetyltransferases in mammals. Bioessays. 2018;40:e1800078. doi: 10.1002/bies.201800078. [DOI] [PubMed] [Google Scholar]
  68. Wang Z., Zang C., Cui K., Schones D.E., Barski A., Peng W., Zhao K. Genome-wide mapping of HATs and HDACs reveals distinct functions in active and inactive genes. Cell. 2009;138:1019–1031. doi: 10.1016/j.cell.2009.06.049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Weinert B.T., Narita T., Satpathy S., Srinivasan B., Hansen B.K., Scholz C., Hamilton W.B., Zucconi B.E., Wang W.W., Liu W.R. Time-resolved analysis reveals rapid dynamics and broad scope of the CBP/p300 acetylome. Cell. 2018;174:231–244.e12. doi: 10.1016/j.cell.2018.04.033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Yang C., Wu J., Zheng Y.G. Function of the active site lysine autoacetylation in Tip60 catalysis. PLoS One. 2012;7:e32886. doi: 10.1371/journal.pone.0032886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Zeng L., Zhang Q., Gerona-Navarro G., Moshkina N., Zhou M.M. Structural basis of site-specific histone recognition by the bromodomains of human coactivators PCAF and CBP/p300. Structure. 2008;16:643–652. doi: 10.1016/j.str.2008.01.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. Zlatanova, Thakar H2A.Z: view from the top. Structure. 2008;16:166–179. doi: 10.1016/j.str.2007.12.008. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Document S1. Transparent Methods, Figures S1–S5, and Tables S2–S4
mmc1.pdf (3.6MB, pdf)
Table S1. List of Peptides in Array 1 and 2, Related to Figures 3 and S3
mmc2.xlsx (28.5KB, xlsx)

Articles from iScience are provided here courtesy of Elsevier

RESOURCES