MafK mediates chromatin remodeling to silence IRF8 expression in non-immune cells in a lineage-specific manner

The regulation of gene expression is a result of a complex interplay between chromatin remodeling, transcription factors (TFs) and signaling molecules. Cell differentiation is accompanied by chromatin remodeling of specific loci to permanently silence genes that are not essential for the differentiated cell activity. The molecular cues that recruit the chromatin remodeling machinery are not well characterized. IRF8 is an immune-cell specific TF, and thus, serves as a model gene to elucidate the molecular mechanisms governing its silencing in non-immune cells. A high-throughput shRNA library screen in IRF8 expression-restrictive cells enabled the identification of MafK as modulator of IRF8 silencing, affecting chromatin architecture. ChIP-seq analysis revealed three MafK binding-regions (-25kb, -20kb and IRF8 6th intron) in the IRF8 locus. These MafK binding-sites are sufficient to repress a reporter-gene when cloned in genome-integrated lentiviral reporter constructs in expression-restrictive cells only, while plasmid-based constructs do not demonstrate such repressive effect. Furthermore, removal of MafK-int6 binding-region from BAC-IRF8 reporter construct was sufficient to promote accessible chromatin conformation. Taken together, we identified and characterized several MafK binding elements within the IRF8 locus that mediate repressive chromatin conformation resulting in the silencing of IRF8 expression only in non-immune cells. MafK ChIP-seq was performed in two cell lines, mouse fibroblast cell-line NIH3T3 and mouse macrophage cell-line RAW. For NIH3T3 cells, 4 IP replicates were analyzed and 1 input sample. For RAW cells 3 IP replicates were analyzed and 1 input sample.