Abstract
Monomethylation of histone H3 lysine 4 (H3K4me1) is enriched at enhancers that are primed for activation and the levels of this histone mark are frequently altered in various human cancers. Yet, how alterations in H3K4me1 are established and the consequences of these epigenetic changes in tumorigenesis are not well understood. Using ChIP-Seq in human colon cancer cells, we demonstrate that mutant p53 depletion results in decreased H3K4me1 levels at active enhancers that reveal a striking colocalization of mutant p53 and the H3K4 monomethyltransferase MLL4 following chronic tumor necrosis factor alpha (TNFα) signaling. We further reveal that mutant p53 forms physiological associations and direct interactions with MLL4 and promotes the enhancer binding of MLL4, which is required for TNFα-inducible H3K4me1 and histone H3 lysine 27 acetylation (H3K27ac) levels, enhancer-derived transcript (eRNA) synthesis, and mutant p53-dependent target gene activation. Complementary in vitro studies with recombinant chromatin and purified proteins demonstrate that binding of the MLL3/4 complex and H3K4me1 deposition is enhanced by mutant p53 and p300-mediated acetylation, which in turn reflects a MLL3/4-dependent enhancement of mutant p53 and p300-dependent transcriptional activation. Collectively, our findings establish a mechanism in which mutant p53 cooperates with MLL4 to regulate aberrant enhancer activity and tumor-promoting gene expression in response to chronic immune signaling.
Highlights
Monomethylation of histone H3 lysine 4 (H3K4me1) is enriched at enhancers that are primed for activation and the levels of this histone mark are frequently altered in various human cancers
We further reveal that mutant p53 forms physiological associations and direct interactions with MLL4 and promotes the enhancer binding of MLL4, which is required for TNF␣-inducible H3K4me1 and histone H3 lysine 27 acetylation (H3K27ac) levels, enhancer-derived transcript synthesis, and mutant p53-dependent target gene activation
We compared our previously published global run-on sequencing (GRO-Seq) data in SW480 cells [39] with the MLL4 and mutp53 ChIP-Seq data and found a striking parallel between the mutp53- and MLL4-binding profiles and GROSeq signals in response to chronic TNF␣ signaling (Fig. 1B). This strong parallel between mutp53 and MLL4 binding at active enhancers in response to chronic TNF␣ signaling is demonstrated by the UCSC Genome Browser tracks of mutp53, MLL4, H3K27ac, and H3K4me1 ChIP-Seq data and GRO-Seq signals at the MMP9 and CCL2 enhancers (Fig. 1C), which are among the signal-dependent enhancers that we recently found are bound and activated by mutp53 and NF-B in response to chronic immune signaling [39]
Summary
P53R273H,P309S and MLL4 colocalize at active enhancers and form physiological associations in colon cancer cells. P53 R273H is required to direct MLL3/4C recruitment on chromatin as revealed by the 2-fold increase in ASH2L binding at the promoter (Fig. 3D, column 4 versus 2), which is consistent with our cell-based ChIP data that revealed a requirement for mutp in regulating MLL3/4 enhancer interactions (Fig. 2C). QRT-PCR analyses revealed an ϳ2-fold reduction in the TNF␣-induced expression levels of both the MMP9 and CCL2 eRNAs following MLL3/4 knockdown (Fig. 5B), which correlated with a decrease in the TNF␣-induced mRNA expression levels of MMP9 and CCL2 (1.8-fold and 11-fold, respectively) Consistent with these changes in enhancer transcription and gene expression, we identified that MLL3/4 depletion results in a notable (ϳ55%) reduction in the TNF␣-induced binding of RNAPII at the enhancer versus nonspecific control regions of MMP9 and CCL2 (Fig. S3B). These findings establish a requirement for MLL4 in regulating TNF␣-induced alterations in enhancer and tumor promoting gene activation that are linked to enhanced cancer cell invasion
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