Local regulation of DNA methylation by the transcription factor REST

Abstract

Transcriptional regulation in eukaryotes is realized through intricate interactions between transcription factors and chromatin. DNA methylation constitutes a chromatin modification that is associated with transcriptional silencing (Deaton and Bird, 2011). Whole-genome methylation profiling in mammals has revealed widespread cytosine methylation with characteristic hypomethylation at cis-regulatory elements. Hypomethylation is typically present within CpG islands and distal CpG-poor regions (Stadler et al., 2011). Previous investigations have shown, that some DNA-binding factors like the RE1-silencing transcription factor (REST) directly reduce methylation at these sites. However, how DNA-binding factors mediate such local methylation changes remains largely unknown. Hence, I studied the regulation of DNA methylation by the transcription factor REST in mouse embryonic stem cells (mESCs). I ectopically expressed different REST mutants and profiled DNA methylation at distal REST binding sites. While the full-length protein is necessary and sufficient to reduce methylation at its binding sites, REST’s DNA-binding domain lacks this ability. Instead, hypomethylation at binding sites required DNA-binding factors with interaction domains. The N-terminal REST mutant for example recruits SIN3A to binding sites and shows strong DNA demethylation ability. These experiments suggest that hypomethylation is not an obligatory consequence of protein binding, but rather requires interaction domains, reflecting the potential involvement of cofactors. I inquired whether TET enzymes contribute to reduced methylation within REST binding sites. Complete Tet1/2/3 deficiency in mouse stem cells caused a strong localized hypermethylation in the immediate vicinity of the REST motif. Whether TET proteins are recruited to REST binding sites through common cofactors or indirect mechanisms remains to be determined. I also characterized chromatin accessibility and nucleosome positioning in the different REST mutant re-expression cells. Interestingly, REST mutants that were competent to decrease DNA methylation also increased chromatin accessibility and nucleosome positioning. This could potentially link the chromatin remodeling ability of transcription factors to hypomethylation around binding sites. In summary, the presented study dissected REST induced methylation patterns around binding sites and described several of its required molecular components. This presents an example for a dynamic interplay between genetic and epigenetic information.