Figure 4: Pcgf6 Directly Regulates Key Pluripotency Factors in ESCs, Including Oct4, Sox2, Lin28b, and Nanog.

(A) Heat map representations showing the chromatin-binding distribution of various epigenetic regulators in CCE ESCs. For Pcgf6, ChIP-seq experiments were performed with two different antibodies, as indicated (Ab#1 and 2; See Methods). ChIP-seq data for H3K4me3, H3K27me3, Suz12, Ezh2, and Rnf2 were obtained from the Bernstein lab³⁸, and data for Rbbp5 and Wdr5 were obtained from the Ihor lab³². Regions decorated with active (H3K4me3), repressive (H3K27me3), and bivalent poised/repressive (both H3K4me3 and H3K27me3) chromatin marks are indicated by the vertical bar at left. (B) Pcgf6 binds promoter regions of key pluripotency genes in ESCs. ChIP-seq data for Pcgf6 (this study) were uploaded to the Integrative Genomics Viewer browser and compared with ChIP-seq data for H3K27me3, Suz12, Ezh2, and Rnf2 (as above) and Oct4, Rbbp5, Wdr5, and Wdr5-FL (Ihor lab³²). The scale of each genomic region is indicated at the top of each panel. The TSSs of Sox2, Oct4, Nanog, Lin28a, Myc, and Lin28b target genes are shown as black squares with the arrow showing transcription direction. Green indicates Pcgf6 binding regions; orange indicates regions with histone modification markers; blue indicates regions bound by Oct4, PRC1, PRC2, and TrxG components. (C) Heat map showing that changes in gene expression caused by Pcgf6 knockdown is recapitulated by knockdown of Pcgf6 targets. CCE ESCs were transfected with siRNAs targeting various Pcgf6-bound genes (named at the top), and RT-qPCR was performed ~24 h later to detect expression of a panel of Pcgf6-bound genes (listed at right). Expression levels were normalized to cells treated with the non-targeting siRNA (Control) and displayed as a heat map. The hierarchical tree was created by Cluster and visualized by Java TreeView. The blue hierarchical lines and yellow rectangle indicate the major cluster. (D) Proposed model for the central role of Pcgf6 in modulating the ESC core circuitry. Pcgf6 activates the key regulators Oct4, Sox2, Nanog, and Lin28 in ESCs in a noncanonical fashion and directly regulates the expression of other factors, including Kpna2, Rfwd3, Nup88, Rpa2, Tmpo, and Snurf, which positively regulate the ESC core circuitry.