Abstract 1513: MLH1 silencing is caused by hypoxia through epigenetic regulation..
Authors: Lu Yuhong , Wajapeyee Narendra , Turker Mitchell S. , Glazer Peter M. 
Published: August 2014

Abstract
Transcriptional silencing of the DNA mismatch repair (MMR) gene, MLH1, is one of the main causes of microsatellite instability in sporadic cancers of the colon and other sites. We and others have shown that hypoxia, a key microenvironmental stress in solid tumors, causes reduced expression of MLH1via transient transcriptional repression mediated by both HIF-dependent and independent pathways. Here, we further investigated whether hypoxia-induced down-regulation of MLH1 is associated with epigenetic modifications that may mark the locus for long-term silencing. We find that hypoxia drives epigenetic repression of the MLH1 promoter with decreased H3K4 methylation as a key repressive modification. We find that this is mediated by the H3K4 demethylases, LSD1 (lysine-specific histone demethylase 1) and PLU-1/JARID1B. In RKO cells containing a reporter construct consisting of the MLH1 promoter driving selectable thymidine kinase (TK) and blasticidin resistance (BlastR) genes, long-term silencing of the MLH1 promoter was produced by exposure of the cells to hypoxia as compared to minimal silencing under normoxia. Importantly, knockdown of LSD1 blocked the hypoxia-induced MLH1 promoter silencing in this reporter system.
In RKO cells, the endogenous MLH1 promoter is constitutively silenced by DNA hypermethylation at CpG sites. MLH1 re-expression can be induced by 5-aza-2-deoxycytidine (5-Aza-dC), a DNA methyltransferase inhibitor, accompanied by increased levels of H3K9 acetylation and H3K4 methylation at it promoter. But the promoter rapidly becomes re-silenced after removal of 5-Aza-dC. However, we find that knockdown of LSD1 or its corepressor, CoRest, blocks MLH1 resilencing in the RKO cells. Consistently, ChIP analyses revealed that the control RKO cells (with a GFP knockdown vector) showed return of both H3K9 acetylation and H3K4 methylation back to pre-treatment levels coupled with MLH1 re-silencing upon removal of 5-Aza-dC, but intriguingly, high levels of H3K9 acetylation and H3K4 methylation were also observed at MLH1 promoters in RKO LSD1sh cells. Analysis of DNA methylation revealed that MLH1 promoter methylation levels were reduced by 5-Aza-dC treatment, as expected. However, after removal of 5-Aza-dC, promoter methylation levels returned back to the pre-treatment levels only in the control RKO cells. In the RKO cells with shRNA mediated knock down of either LSD1 or CoRest, the promoter re-methylation process was blocked. Collectively, the results demonstrate that the LSD1/CoRest repressive complex is essential for the MLH1 silencing and that hypoxia is a critical driving force for stable transcriptional silencing of MLH1 through a concerted pattern of chromatin and DNA modifications, thereby promoting genetic instability and tumor progression.
Citation Format: Yuhong Lu, Narendra Wajapeyee, Mitchell S. Turker, Peter M. Glazer. MLH1 silencing is caused by hypoxia through epigenetic regulation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1513. doi:10.1158/1538-7445.AM2013-1513