29108

PYCARD

ASC|CARD5|TMS|TMS-1|TMS1;PYD and CARD domain containing;PYCARD;PYD and CARD domain containing

apoptosis-associated speck-like protein containing a CARD|caspase recruitment domain-containing protein 5|target of methylation-induced silencing 1

16p11.2

protein-coding

DNA methylation and histone modifications have emerged as key mechanisms in transcriptional regulation. The target of methylation-induced silencing 1 (TMS1) is a bipartite protein. Recent studies have indicated that methylation-associated silencing of TMS1 occurs in many cancers. However, whether and how TMS1 is regulated by epigenetic mechanisms in cancers remains unknown. In this study we showed that methylation of the TMS1 promoter occurred in five of six hepatocellular carcinoma (HCC) cell lines. TMS1 expression was reduced in four HCC cell lines and correlated with methylation status. Furthermore, the TMS1 promoter was completely methylated and mRNA expression was undetectable. TMS1 expression could be restored by 5-aza-2-deoxycitidine (5-Aza-dC) (a DNA methyltransferase inhibitor) or trichostatin A (TSA) (a histone deacetylase inhibitor) alone and the promoter methylation was partially reversible. TSA was more efficient than 5-Aza-dC in inducing TMS1 expression, and the combination of 5-Aza-dC and TSA resulted in markedly synergistic reactivation of the gene and completely reversed promoter methylation. Interestingly, TMS1 promoter methylation-associated gene silencing was accompanied by histone H3 Lysine 9 (H3K9) hypoacetylation and trimethylation. 5-Aza-dC and/or TSA also had some effect on conversion of methylated to acetylated H3K9 in restoring TMS1. This conversion was dynamic at the TMS1 promoter and a decrease in H3K9 trimethylation preceded an increase in H3K9 acetylation after 5-Aza-dC and/or TSA treatment. Our results thus suggest that epigenetic inactivation of TMS1 expression is regulated by promoter hypermethylation and H3K9 modifications in a coordinated way.

ASC-2, a multifunctional coactivator, forms a steady-state complex, named ASCOM (for ASC-2 COMplex), that contains the histone H3-lysine-4 (H3K4)-methyltransferase MLL3 or its paralogue MLL4. Somewhat surprisingly, given prior indications of redundancy between MLL3 and MLL4, targeted inactivation of the MLL3 H3K4-methylation activity in mice is found to result in ureter epithelial tumors. Interestingly, this phenotype is exacerbated in a p53(+/-) background and the tumorigenic cells are heavily immunostained for gammaH2AX, indicating a contribution of MLL3 to the DNA damage response pathway through p53. Consistent with the in vivo observations, and the demonstration of a direct interaction between p53 and ASCOM, cell-based assays have revealed that ASCOM, through ASC-2 and MLL3/4, acts as a p53 coactivator and is required for H3K4-trimethyation and expression of endogenous p53-target genes in response to the DNA damaging agent doxorubicin. In support of redundant functions for MLL3 and MLL4 for some events, siRNA-mediated down-regulation of both MLL3 and MLL4 is required to suppress doxorubicin-inducible expression of several p53-target genes. Importantly, this study identifies a specific H3K4 methytransferase complex, ASCOM, as a physiologically relevant coactivator for p53 and implicates ASCOM in the p53 tumor suppression pathway in vivo.