The recent finding that the release of paused initiated Pol II into productive elongation is an important regulatory step at thousands of genes has garnered great interest in the mechanism by which Pol II pausing is regulated (19
). Here, we demonstrate that H4K16Ac and H4K20me3 antagonistically control gene expression by regulating Pol II promoter-proximal pausing. We find that hMOF-mediated H4K16Ac stimulates the release of Pol II from promoter-proximal pausing into productive elongation and is required for the association of the BRD4 and pTEFb complex with chromatin at the TMS1
locus. Methylation of CpG island DNA and the shift to H3K9me2 that accompanies the epigenetic silencing of certain tumor suppressor genes block Pol II access to the embedded TMS1
promoter. However, even in the absence of these factors, repression is maintained by SUV420H2-mediated H4K20me3, which inhibits the recruitment of hMOF and the subsequent acetylation of H4K16 and, in so doing, enforces Pol II pausing (). Whereas CpG island methylation also blocks Pol II promoter access at other silenced genes, those lacking H4K20me3 (e.g., ESR1
) do not exhibit Pol II pausing upon DNA demethylation. We further find that H4K20me3 selectively marks other genes (e.g., JUND
) independently of DNA methylation where it similarly imposes a block to Pol II elongation through a mechanism that involves the local inhibition of H4K16Ac. This is the first study to demonstrate the negative interplay between H4K16Ac and H4K20me3 in the local modulation of Pol II pausing and to show that DNA methylation/H3K9me2 and H4K20me3 act as independent layers to maintain transcriptional repression at certain epigenetically silenced tumor suppressor genes in cancer.
Fig. 9. Model for the multilayered regulation of TMS1 gene repression and Pol II promoter-proximal pausing by DNA methylation and histone H4 modifications. (1) In the stably repressed state in which the CpG island is methylated and the locus is associated with (more ...)
We show that H4K16Ac promotes the escape of Pol II from pausing by mediating the recruitment of the bromodomain protein BRD4 and the pTEFb complex. Our results are consistent with those of Zippo et al. (69
), who showed that hMOF-mediated H4K16Ac at a distal enhancer relieves Pol II pausing at the FOS1
promoter. In that case, hMOF was selectively recruited to the enhancer by 14-3-3ε- and 14-3-3ζ-mediated binding to histone H3 phosphorylated at S10 (H3S10p). hMOF is distributed throughout the TMS1
promoter and into the gene body, as it is at many other active genes, suggesting that there may be multiple mechanisms of hMOF targeting (29
) (). hMOF has been shown to exist as part of several different complexes in human cells, including the MSL, MSL1v1, MLL-WDR5, and NSL complexes (5
). We previously showed that MSL1, a component of the MSL complex, colocalizes with hMOF at the TMS1
locus and that the effect of MSL1 knockdown on H4K16Ac and full-length TMS1
transcript levels parallels that observed upon downregulation of hMOF, suggesting that hMOF acts as part of the MSL complex in this context (29
). Although the precise mechanism of MSL targeting to the TMS1
locus is currently unknown, our data show that this recruitment is inhibited, either directly or indirectly, by SUV420H2 and/or SUV420H2-mediated H4K20me3. Interestingly, hMOF localization and H4K16Ac levels are reestablished throughout the TMS1
locus in the absence of H4K20me3 even though SUV420H2 and H4K20me3 are normally localized to a single sharp peak near the promoter (). Similarly, H4K16Ac is distributed throughout the JUND
locus in the absence of H4K20me3 even though H4K20me3 is enriched mainly at the promoter region (). This suggests that the recruitment of the MSL complex/hMOF to the promoter region is critical but that once the MSL complex gains access, it can modify nucleosomes throughout the gene body. Together, these studies point to the potential for multiple histone modifications to control promoter-proximal Pol II pausing.
We found that the promoter region of the unmethylated TMS1
gene is occupied by the NELF complex, regardless of H4K16Ac or levels of gene expression. Considering the role of NELF as a negative elongation factor, the finding that NELF is also present at actively transcribed genes seems paradoxical. Nevertheless, recent work by Gilchrist et al. (18
) showed that silencing of NELF results in both the upregulation and downregulation of target genes. Interestingly, genes that were downregulated in the absence of NELF showed a decrease in H3K4me3 and increased nucleosome occupancy at their promoters (18
). Downregulation of NELF also led to a selective decrease in Pol II levels at the 5′ end of target genes (18
). These data suggest that in addition to its function in enforcing Pol II pausing, NELF also plays a positive role in transcription by maintaining a permissive chromatin conformation and stabilizing the association of Pol II with target gene promoters.
The maintenance of Pol II occupancy, whether paused or actively elongating, may be one mechanism that prevents DNA methylation from encroaching on CpG island promoters. Recent work has shown that resistance of CpG islands to aberrant DNA methylation in human cancer cells is better correlated with Pol II occupancy than with absolute transcript levels (57
). Similarly, recent work from our lab indicates that the propensity for genes to undergo remethylation after DAC-induced demethylation is inversely correlated with the long-term maintenance of Pol II occupancy (28
). Pol II occupancy may prevent DNA methylation directly or indirectly by promoting an open chromatin structure rich in active histone marks, such as methylated H3K4, which has been shown to block the interaction of de novo
DNA methyltransferases with nucleosomes (26
). The enforcement of Pol II pausing may be a step that precedes DNA methylation in the aberrant silencing of tumor suppressor genes in cancers. Subsequent dissociation of Pol II—for example, by events that dysregulate NELF or other factors involved in the maintenance of paused Pol II (e.g., H4K20me3)—may allow for abnormal DNA methylation and stable gene repression. In this regard, it is noteworthy that many of the factors that control Pol II pausing are also dysregulated in human cancers. Cancer cells exhibit a global loss of H4K16Ac and H4K20me3 relative to normal cells (16
). H4K16Ac and hMOF levels have been shown to be decreased in primary medulloblastomas and breast cancers (48
). Likewise, H4K20me3 and SUV420H2 are downregulated in human lung and liver tumors (6
is amplified in breast cancer and translocated in NUT midline carcinomas (17
). Crawford et al. (12
) showed that ectopic expression of BRD4
in breast cancer cells suppresses tumor growth and metastasis in vivo
and identified a BRD4-activated gene signature that can predict prognosis in human breast cancer patients.
H4K20me3 is enriched in areas of constitutive heterochromatin, and its depletion results in destabilization of pericentric heterochromatin, compromised telomere integrity, and ectopic activation of endogenous retroelements, supporting the widely held notion that the primary function of H4K20me3 is to stabilize heterochromatin structure (3
). However, recent studies have suggested that H4K20me3 also marks individual genes (9
), but its function, if any, at such loci has not been elucidated. We provide evidence here that SUV420H2 and H4K20me3 are selectively targeted to individual genes and, moreover, that H4K20me3 plays an important role in gene repression through the local inhibition of H4K16Ac. Early work showing that H4K16 acetylation and H4K20 methylation are mutually exclusive on the X chromosome in Drosophila
males and that histone H4 peptides premethylated at H4K20 are poor substrates for p300-mediated H4K16 acetylation led to the suggestion that H4K20me3 and H4K16Ac may be competitive in vivo
). However, top-down mass spectrometry studies have shown that H4K16Ac and H4K20me3 can coexist on the same histone H4 tail, and additional studies indicate that depletion of SUV420H has no impact on global levels of H4K16Ac, favoring a model in which the two modifications are independently regulated (66
). Our data clearly show that SUV420H2 and/or H4K20me3 antagonizes H4K16Ac in vivo
and that this derives from the local inhibition of hMOF and MSL complex recruitment. While our data cannot distinguish whether it is the presence of the H4K20me3 mark itself or another function of SUV420H2 (i.e., methylation of nonhistone targets and/or scaffolding functions) that blocks MSL recruitment, the finding that SUV420H2 knockdown affected H4K16Ac, Pol II dynamics, and gene expression only at genes marked by H4K20me3 but not at genes lacking H4K20me3 (ESR1
) speaks to a gene-specific regulatory mechanism wherein the local targeting of SUV420H2 and deposition of H4K20me3 itself are critical.
Current models suggest that H4K20 methylation occurs in a progressive stepwise process wherein H4K20 monomethylated by the histone methyltransferase PR-SET7 serves as a substrate for di- and trimethylation by SUV420H (47
). The vast majority of H4K20 in human cells exists in the H4K20me2 form; the mechanism(s) that regulates the localized conversion of a small fraction into H4K20me3 is currently unknown (66
). Indeed, recent biochemical and structural data suggest that the chromoshadow domain of the MSL3 component of the MSL complex can bind H4K20me1/2 but cannot bind H4K20me3 (30
). Thus, the local conversion to H4K20me3 by SUV420H2 could potentially block MSL complex recruitment by precluding MSL3 binding. Other studies show that HP1 binds to SUV420H1/2 and is necessary for the maintenance of H4K20me3 at constitutive heterochromatin and at imprint control regions and that depletion of H3K9me3 leads to a loss of H4K20me3 at major satellite repeats and endogenous retroviruses (35
), suggesting that SUV420H (and hence H4K20me3) may be directed to these regions by an HP1-mediated association with H3K9me3 (50
). Indeed, depletion of H3K9me3 leads to a loss of H4K20me3 at major satellite repeats and endogenous retroviruses (35
). SUV420H2 has also been shown to interact with RB1, and H4K20me3 levels are decreased at telomeric and pericentric heterochromatin in RB1-deficient cells (20
). Whether HP1 or RB1 plays a similar role in SUV420H2 targeting to individual genes remains to be determined.
Trimethylation of H4K20 at the TMS1
locus is likely an acquired event in cancer cells, and the subsequent repression is a prerequisite to the more stable and heritable silencing imposed by DNA methylation. In the absence of DNA methylation, inhibition of H4K20me3 allows for the rerecruitment of hMOF to the locus, reestablishment of H4K16Ac and TMS1
upregulation, indicating that the initial silencing was due not to a loss of hMOF expression or activity but rather to the directed repression by SUV420H2 and/or H4K20me3. Indeed, H4K20me3 is absent from the TMS1
locus in normal fibroblasts and immortalized breast epithelial cells (29
) (data not shown), and whereas downregulation of hMOF in MCF7 cells leads to depletion of H4K16Ac and TMS1
repression, it is not sufficient to evoke H4K20me3 (29
). Thus, it is likely that other trans
-acting factors present in MDA-MB231 cells target SUV420H2 to the TMS1
locus. Although the mechanisms triggering the subsequent acquisition of H3K9me2 and aberrant DNA methylation at TMS1
are presently unknown, our data suggest that once this is achieved, repression can be maintained independently of H4K20me3. Indeed, the finding that other genes are marked by H4K20me3 in a cancer cell line independently of DNA methylation (e.g., JUND
), where it similarly enforces Pol II pausing, suggests that the targeting of H4K20me3 is both gene and cell type specific, and whereas this event may put certain genes at risk of subsequent DNA methylation, it is not sufficient to drive this process.
We found that depletion of DNA methylation at TMS1 led to a concomitant inhibition of H3K9me2 and the reacquisition of H3K4me2 and Pol II but had no impact on H4K20me3. Similarly, whereas depletion of H4K20me3 allowed for restoration of H4K16Ac, it had no effect on DNA methylation or H3K9me2. These data indicate that once established, DNA methylation and H3K9me2 are coordinately regulated but are independent of H4K20me3 and that the two represent independent layers of gene repression. DNA methylation (and the linked H3K9me2) is dominant in this regard, in that depletion of H4K20me3 cannot overcome the block to Pol II access imposed by DNA methylation (). These data support a model in which DNA methylation serves as the final lock at tumor suppressor loci to mediate stable and heritable repression.
These findings have important implications for the application of “epigenetic therapy” as an approach to cancer treatment. DAC (Decitabine) and the related agent 5-azacytidine (Vidaza) are currently being used in the treatment of myelodysplastic syndrome (MDS) and acute myeloid leukemia (23
). Whereas both cell culture experiments and clinical studies have shown that DAC treatment leads to a global demethylation of DNA and reactivation of some silenced genes, there is a propensity for remethylation and resilencing after drug removal (15
). This has been attributed to the residual presence of repressive marks, such as H3K9me3 and H3K27me2/3, which are unaffected by DNA demethylation, and the failure to regain active features (such as H3K79me2) (24
). We show here that H4K20me3 is similarly unaffected by DNA demethylation and that the residual presence of H4K20me3 maintains gene repression by enforcing Pol II promoter-proximal pausing. The combined inhibition of DNA methylation and H4K20 methylation leads to a synergistic reactivation of TMS1
gene expression and recapitulates a chromatin environment similar to that observed in cells that actively express TMS1
. Thus, combined approaches targeting both DNA methylation and histone methylation may be therapeutically beneficial and warrant consideration in the development of novel epigenetic therapy approaches.