MLH1 Silencing is Caused by Hypoxia Through Epigenetic Regulation

Assoc Rsrch Scientist Ther Radiology
15 York St, New Haven, CT 06510-3221

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 have shown that hypoxia, a key microenvironmental stress in solid tumors, causes reduced expression of MLH1 via  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 thatmay 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.