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. 2023 Oct 27;51(19):10194-10217.
doi: 10.1093/nar/gkad700.

HIRA-mediated loading of histone variant H3.3 controls androgen-induced transcription by regulation of AR/BRD4 complex assembly at enhancers

Viacheslav M Morozov  1 , Alberto Riva  2 , Sadia Sarwar  1 , Wan-Ju Kim  1 , Jianping Li  3 , Lei Zhou  4   5 , Jonathan D Licht  3   5 , Yehia Daaka  1   5 , Alexander M Ishov  1   5
Affiliations

HIRA-mediated loading of histone variant H3.3 controls androgen-induced transcription by regulation of AR/BRD4 complex assembly at enhancers

Viacheslav M Morozov et al. Nucleic Acids Res. .

Abstract

Incorporation of histone variant H3.3 comprises active territories of chromatin. Exploring the function of H3.3 in prostate cancer (PC), we found that knockout (KO) of H3.3 chaperone HIRA suppresses PC growth in vitro and in xenograft settings, deregulates androgen-induced gene expression and alters androgen receptor (AR) binding within enhancers of target genes. H3.3 affects transcription in multiple ways, including activation of p300 by phosphorylated H3.3 at Ser-31 (H3.3S31Ph), which results in H3K27 acetylation (H3K27Ac) at enhancers. In turn, H3K27Ac recruits bromodomain protein BRD4 for enhancer-promoter interaction and transcription activation. We observed that HIRA KO reduces H3.3 incorporation, diminishes H3.3S31Ph and H3K27Ac, modifies recruitment of BRD4. These results suggest that H3.3-enriched enhancer chromatin serves as a platform for H3K27Ac-mediated BRD4 recruitment, which interacts with and retains AR at enhancers, resulting in transcription reprogramming. In addition, HIRA KO deregulates glucocorticoid- (GR) driven transcription of genes co-regulated by AR and GR, suggesting a common H3.3/HIRA-dependent mechanism of nuclear receptors function. Expression of HIRA complex proteins is increased in PC compared with normal prostate tissue, especially in high-risk PC groups, and is associated with a negative prognosis. Collectively, our results demonstrate function of HIRA-dependent H3.3 pathway in regulation of nuclear receptors activity.

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Figures

Graphical Abstract
Graphical Abstract
Figure 1.
Figure 1.
Modification of HIRA affects proliferation of PC cells. (A) Proliferation of R1-AD1 and Daxx KO, AR KO, HIRA KO cell lines. (B) colony formation assays with these cell lines in androgen deprived (top) and R1881 stimulated conditions (bottom). Left: representative images; middle: average colony size; right: combined colonies area calculated with ImageJ. P-values: **** < 0.0001. (C) Modification of HIRA affects xenograft growth of PC cells. 106 R1-AD1 cells (CTL), Daxx KO, AR KO and HIRA KO cells were implanted subcutaneously in athymic nude mice, 10 per group. 4 weeks later, tumors were collected and photographed (left). Tumor weight in g (right).
Figure 2.
Figure 2.
Differential gene expression after androgen stimulation. Expression analysis (RNA-seq, volcano plot) of R1-AD1 cells, parental (Control), AR KO, Daxx KO and HIRA KO. Numbers correspond to genes that are at least 2-fold (P-adjusted < 0.05) up- (red) or downregulated (green) by 4 h (top row), 12 h (middle row) or 24 h (bottom row) of R1881 stimulation compared with androgen-deprived (72 h) in parental cells. X: expression, log2; Y: P-value, log10. See Supplementary Figure S2 for Venn diagrams and regulated pathways analysis.
Figure 3.
Figure 3.
H3.3 association with TSS of androgen-regulated genes; analysis within cell lines. ChIP-seq profiles of H3.3 (endogenous HA-H3.3) at the 409 androgen-up- (left) and 328 androgen-downregulated (right) genes in R1-AD1 parental (Control), AR KO, Daxx KO and HIRA KO cells in the androgen-deprived (0h) and androgen-induced conditions (at 4, 12, 24 h). 0: TSS. See Supplementary Figure S3 for time points comparison.
Figure 4.
Figure 4.
H3K27Ac association with TSS of androgen-regulated genes; analysis within cell lines. ChIP-seq profiles of H3K27Ac at the 409 androgen-up- (left) and 328 androgen-downregulated (right) genes in R1-AD1 parental (Control), AR KO, Daxx KO and HIRA KO cells in the androgen-deprived (0 h) and androgen-induced conditions (at 4, 12, 24 h). 0: TSS. See Supplementary Figure S5 for time points comparison.
Figure 5.
Figure 5.
AR association with chromatin is regulated by HIRA. Left: Metaplot of AR peaks (position ‘0’ at 4 h in R1-AD1 parental cells) in R1-AD1 parental, HIRA KO and Daxx KO in androgen-deprived (72 h, red) and R1881 stimulated for 4 h (green), 12 h (blue), 24 h (yellow). In parental cells, AR association with chromatin is elevated at 4 h of stimulation and remains at the same levels at later time points. In HIRA KO, association is reduced in deprived condition, is much higher compared with parental at 4h, and next is substantially reduced at 12 and 24 h. Analysis of AR at enhancers nearest to the 409 up- (middle) and 328 downregulated (right) genes is similar to the overall AR behavior. See Supplementary Figure S7 for time points comparison.
Figure 6.
Figure 6.
Dynamics of H3.3 at enhancers. Metaplot of H3.3 (endogenous HA-H3.3) at AR peaks (position ‘0’: AR at 4 h in R1-AD1 parental cells) at enhancers associated with up- (left) and down-regulated (right) genes in R1-AD1 parental (Control), AR KO, Daxx KO and HIRA KO in androgen-deprived (72 h, red) and R1881 stimulated for 4 h (green), 12 h (blue), 24 h (yellow). Levels of H3.3 at AR-positive enhancers associated with upregulated genes are elevated at 24 h of induction in parental and Daxx KO cells. H3.3 is reduced at enhancers in AR KO cells, suggesting AR function in maintenance of this transcription associated histone variant, and HIRA KO, confirming HIRA chaperone function. See Supplementary Figure S9 for time points comparison.
Figure 7.
Figure 7.
Dynamics of H3K27Ac at enhancers. Metaplot of H3K27Ac ChIP-seq analysis at AR peaks (position ‘0’: AR at 4 h in R1-AD1 parental cells) at enhancers associated with up- (left) and down-regulated (right) genes in R1-AD1 parental (Control), AR KO, Daxx KO and HIRA KO in androgen-deprived (72 h, red) and R1881 stimulated for 4 h (green), 12 h (blue), 24 h (yellow). In parental cells, H3K27Ac is gradually elevated during androgen treatment, to higher levels at enhancers associated with up-regulated genes. AR KO completely abolished, and HIRA KO strongly reduced accumulation of H3K27Ac at enhancers associated with both up- and downregulated genes. See Supplementary Figure S10 for time points comparison.
Figure 8.
Figure 8.
Dynamics of H3.3S31Ph at enhancers; analysis within cell lines. Metaplot of H3.3S31Ph ChIP-seq analysis at AR peaks (position ‘0’: AR at 4 h in R1-AD1 parental cells) at enhancers associated with up- (left) and down-regulated (right) genes in R1-AD1 parental (Control), AR KO, Daxx KO and HIRA KO in androgen-deprived (72 h, red) and R1881 stimulated for 4 h (green), 12 h (blue). Levels of H3.3S31Ph are androgen-induced in parental and Daxx KO cells, no changes in AR KO cells, and are reduced in HIRA KO cells, resembling H3.3 dynamics. See Supplementary Figure S11 for time points comparison.
Figure 9.
Figure 9.
Dynamics of BRD4 at enhancers. Metaplot of BRD4 ChIP-seq analysis at AR peaks (position ‘0’: AR at 4 h in R1-AD1 parental cells) at enhancers associated with up- (left) and down-regulated (right) genes in R1-AD1 parental (Control), AR KO, Daxx KO and HIRA KO in androgen-deprived (72 h, red) and R1881 stimulated for 4 h (green), 12 h (blue). BRD4 accumulated within enhancers at AR binding sites in parental cells, with dynamics mirroring those of AR (Figure 2). AR KO abolished BRD4 accumulation. In HIRA KO cells, BRD4 dynamics is similar to AR, first accumulating at 4h of stimulation and next reducing at 12 h. See Supplementary Figure S12 for time points comparison.
Figure 10.
Figure 10.
DNA accessibility analyzed of AR-positive enhancers by ATAC-seq. Metaplot of DNA accessibility at AR peaks (position ‘0’: AR at 4 h in R1-AD1 parental cells) at enhancers associated with up- (left) and down-regulated (right) genes in R1-AD1 parental (Control), Daxx KO and HIRA KO in androgen-deprived (72 h, red) and R1881 stimulated for 4 h (green). Stimulation for 4 h increased DNA accessibility at AR binding sites within enhancers associated with up- (to the higher extend) and downregulated genes, in all but AR KO cells; HIRA KO reduces DNA accessibility. See Supplementary Figure S13 for time points comparison.
Figure 11.
Figure 11.
FKBP5 gene and SE are regulated by AR, GR and HIRA. (A) Expression of FKBP5. R1-AD1 cells (parental, AR KO, Daxx KO and HIRA KO) were androgen-deprived for 72 h (control), induced with 1 nM of R1881 for 4, 12, 24 h and subjected by FKBP5 RNA analysis at three bioreplicates (left) and western blot analysis with corresponding antibodies (right). AR KO and HIRA KO substantially reduced induction of FKBP5 at RNA and protein levels. (B) Epigenetic profiling of FKBP5 SE. R1-AD1 cells (parental, AR KO, Daxx KO, HIRA KO) were androgen-deprived for 72 h (Control) and induced with 1 nM of R1881 for 4, 12, 24 h. H3K4me1 (enhancer), H3K27Ac (active enhancer), H3.3, H3.3S31Ph, AR and BRD4 profiling were analyzed by ChIP-seq. DNA accessibility analyzed by ATAC-seq. (C) HIRA KO abolishes induction of FKBP5 gene by glucocorticoid receptor (GR). R1-AD1 cells (parental, AR KO, Daxx KO, HIRA KO) were hormone-deprived for 72 h (0), induced with indicated concentrations of dexamethasone (Dex) for indicated time, and analyzed by western blot with corresponding antibodies. AR KO elevates GR levels; AR and HIRA KO substantially reduced induction of FKBP5 protein levels by Dex. Actin: loading control.
Figure 12.
Figure 12.
Model of HIRA/H3.3 function in AR transcription regulation. Control cells. (I) HIRA chaperone complex recruits H3.3 at enhancers (1). CHK1 and IKKa phosphorylates H3.3 at S31 (S31ph; 2), that activates p300 (3) for acetylation of H3K27 (4). (II) androgen treatment recruits AR to ARE that interacts with and co-recruits bromodomain protein BRD4 and displaces nucleosome at ARE (5); BRD4 binds to H3K27Ac at enhancer (6). (III) release of BRD4 from AR opens AR-NTD for recruitment of p300 (7) that is activated by increased levels S31ph (8, similar to 3). (IV) active p300 further acetylates H3K27 (9, similar to 4), resulted in additional binding of BRD4 to H3K27Ac at enhancers (10, similar to 6), recruitment of co-activators (11) and formation of completed transcription complex (pink cloud), enhancer-promoter contact and transcription activation (12). Steps 6 and 7 are speculative and thus highlighted in red. Model does not reflect several details and complementary scenarios, including but not limiting: effect of AR-dimer, co-recruitment of p300 and AR co-regulators, transfer of PolII from enhancer to promoter, BRD4-mediated transcription elongation. HIRA-KO cells. (I) No H3.3 recruitment, reduced H3K27Ac. (II) androgen induction recruits AR that interacts with and co-recruits BRD4 at AREs (1). (III) BRD4 cannot bind to chromatin due to reduced H3K27Ac, thus accumulating together with AR at AREs, and blocking recruitment of AR co-regulators (2). (IV) in the absence of co-regulators and reduced BRD4 binding to H3K27Ac at enhancers, AR and BRD4 have decreased retention at AREs (3); AR transcription complex does not form, and transcription regulation is weaker compared to parental cells.
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