TRIM28 and novel interacting KRAB-ZNFs preserve self-renewal of human pluripotent stem cells through H3K9me3 and DNA methylation mediated repression of pro-differentiation genes

Reprogramming to induced pluripotent stem cells (iPSCs) and differentiation of pluripotent stem cells (PSCs) are primarily regulated by epigenetic machinery. Tripartite motif protein 28 (TRIM28), a universal mediator of Krüppel-associated box domain zinc fingers (KRAB-ZNFs), is known to regulate both processes, however exact mechanism and identity of participating KRAB-ZNF genes remains unknown. Here, using a novel reporter system, we first showed that TRIM28/KRAB-ZNFs alter DNA methylation patterns in addition to H3K9me3 to cause gene repression during reprogramming. Next, using several expression datasets, we identified novel KRAB-ZNFs (ZNF114, ZNF483 and ZNF589) in the human genome that regulate pluripotency. Mechanistically, we demonstrated that these KRAB-ZNFs directly alter gene expression of important developmental genes by modulation of H3K9me3 and DNA methylation on their promoters. In summary, TRIM28 employs novel KRAB-ZNFs to evoke epigenetic silencing of its target differentiation genes by H3K9me3 and DNA methylation. Identification of the targets repressed by pluripotency-specific KRAB-ZNFs. ChIP-seq analysis focused on genome-wide binding profile of the pluripotency-specific KRAB-ZNFs (ZFP57, ZNF114, ZNF483 and ZNF589). RNA-seq analysis included three experiments: comparison of the gene expression profile between fibroblasts and induced pluripotent stem cells derived from them, analysis of gene expression changes during reprogramming of the fibroblasts with and without TRIM28 knockdown and analysis of gene expression changes in hESCs and iPSCs upon trim28 knockdown. In the latter analysis, pluripotent stem cells cultured without FGF to trigger differentiation were also tested. ------------------------------------ This represents the ChIP-seq component only.