Gene Regulation In The Immune System

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

IRF8 shapes epigenome landscape in adult microglia and directs the transcriptome program. We examined microglia from Irf8 KO mice and found gross abnormal in morphology, lacking ramified extensions. Bulk and single cell (sc) RNA-seq analyses revealed many genes that make up characteristic features of microglia, e.g., Cx3cr1, Iba1, Tyrobp were downregulated in Irf8 KO cells. Further, many Alzheimer’s disease associated microglia (DAM) genes were dysregulated in Irf8 KO microglia, along with dysregulation of ISGs and lysosomal genes. A salient feature was that the transcription factors, Sall1 and Batf3, required for development of microglia was also downregulated in IRF8 KO microglia, illustrating that IRF8 directs a microglia transcription factor cascade to establish microglia identity. Using a modified Cut&Run method, we determined IRF8 binding sites in the adult microglia genome, which provided a mechanistic insight into IRF8 action. Our results demonstrated that (a) overall IRF8 distribution patterns are quite different from those in peripheral monocytes and their progenitors, although IRF8 binding motifs were similar to those in peripheral myeloid cells, i.e., the ETS/IRF composite and ISRE like elements.(b) IRF8 binding sites were enriched in the distant enhancer regions that regulate expression of microglia specific genes. Some of IRF8 bound sites were within the large super-enhancers coinciding with high histone H3K27ac marks. Moreover, many IRF8 bound sites were near the accessible chromatin sites, as identified by ATAC-seq. Thus, Irf8 KO microglia lacked chromatin accessible sites present in WT cells, correlating with the loss of the expression of microglia identity genes. We also caried out global bisulfite DNA methylome analysis and found extensive alterations of the distribution of methylated CpG islands in IRF8 KO microglia. These results show thatIRF8 confers proper transcription factor access and regulates DNA methylation mediated gene silencing. Together, IRF8 shapes epigenome landscape to establish transcriptome program in adult microglia.