Fig 1: POGZ modulates enhancer activities. A Metaplot showing H3K4me3 ChIP-seq density at active enhancer regions in the presence and absence of POGZ. B Metaplot showing H3K4me3 ChIP-seq density at poised enhancer regions in the presence and absence of POGZ. C Up: Metaplot showing H3K27ac ChIP-seq density at active enhancer regions in the presence and absence of POGZ. Bottom: Heat map showing H3K27ac ChIP signals at active enhancers. D Up: Metaplot showing H3K27ac ChIP-seq density around TSS regions in the presence and absence of POGZ. Bottom: Heat map showing H3K27ac ChIP signals around TSS regions. E Snapshots showing H3K27ac ChIP-seq peaks at the indicated genes in control and Pogz−/− ESC-derived neurospheres. Orange: enhancers; Gray: promoters. F ChIP-PCR results showing H3K27ac enrichment at enhancer regions of the indicated neural genes in day 5 control and Pogz−/− ESC-derived neurospheres. ChIP were repeated two times
Fig 2: POGZ and ADNP form a nuclear complex with HP1γ(A) Western blots for POGZ and ADNP in IPs for HP1α and HP1γ from nuclear extracts of E13.5 wild-type mouse cortex(B) Heatmap of POGZ, ADNP, and HP1γ C&R reads across consensus peaks for all three antibodies in E13.5 mouse forebrain, n = 2.(C) POGZ,ADNP, and HP1γ C&R signal overPOGZ C&R peaks in three different segments of the genome: proximal(within 100kb) to downregulated genes (q-value < 0.01), proximal to upregulated genes (q-value < 0.01), proximal to unchanged genes.(D) Heatmap of POGZ, ADNP, and HP1γ C&R reads across consensus peaks proximal (within 100 kb) to Pogz−/− DE genes.(E) Proposed model for POGZ, ADNP, and HP1γ activity at REs. High levels of binding of all three proteins leads to repression of proximal genes (observed upregulation in Pogz−/−), while high levels of POGZ and reduced levels of ADNP and HP1γ leads to activation of proximal genes (and observed downregulation in Pogz−/−).
Fig 3: POGZ promotes ESC neural induction. A Morphology of day 1, 3, and 6 EBs from control and early passage Mut1 ESCs. B Time course qRT-PCR analysis of indicated genes. C Volcano plot showing the up- and down-regulated genes (fold change > 2) of day 6 EBs from control and Pogz−/− ESCs. D Heat map showing the expression of indicated neural genes. Three replicates are shown: KO for Pogz−/− ESCs, and Ctr for control ESCs. E GO analysis of DEGs of day 6 EBs from control and Pogz−/− ESCs. F Morphology of day 2, 4, 7 neurospheres from control-, Pogz−/−, and POGZ-restoring ESCs. G Morphology of day 16 neuronal cells from control-, Pogz−/−, and POGZ-restoring ESCs. White arrows pointing to the neuronal fibers. H Time course qRT-PCR analysis of indicated neural genes during control and Mut1 ESC directional differentiation toward neural progenitors. I Flow showing the percentage of PAX6-positive cells in control and Pogz−/− ESC-derived neurospheres. J IF results showing PAX6-positive cells in day 7 neural progenitors from control, Mut1, and Mut2 ESCs. Bar: 25 μm. K Quantitation of (J) by Image J. Flow and IF experiments were repeated at least two times. qRT-PCR was repeated at least three times
Fig 4: POGZ directly activates and represses target genes. A Pie chart showing the distribution of POGZ binding sites genome wide. B Heat map of POGZ CUT&Tag enrichment in a 6 kb window around the TSS. C Metaplot of POGZ, H3K4me1 and H3K27ac enrichment (normalized per million mapped reads) on ± 3 kb of genes that were bound by POGZ (27,343 peaks). D Metaplot of POGZ, H3K4me1 and H3K27me3 enrichment (normalized per million mapped reads) on ± 3 kb of genes that were bound by POGZ (1808 peaks). E KEGG analysis of POGZ-bound genes in ESCs. F Pie chart showing the overlap of POGZ targets and up-regulated genes. G Overlap of POGZ targets and down-regulated genes. H The enrichment of KLFs and OCT4/SOX2 binding motifs at POGZ-bound loci, as revealed by HOMER. I CUT&Tag and ChIP-seq snapshots showing the co-localization of POGZ, NANOG and OCT4 at the indicated loci. Gray: proximal TSS; Orange: distal regions. J Heat map of NANOG, OCT4 and POGZ signals across sites bound by POGZ (n = 11,000). Each row represents a 6 kb window centered on the peak midpoint. K Metaplot of POGZ, MED1, ESRRB and KLF4 enrichment (normalized per million mapped reads) on ± 2 kb window across all 231 super-enhancers that have been identified in ESCs by Whyte [41]. L Double IF staining of NANOG and FLAG-POGZ in ESCs. Bar: 25 μm. M IP results showing that OCT4 interacts with POGZ in ESCs. Star pointing to the IgG heavy chain. All IF and WB experiments were repeated at least two times
Fig 5: Neuronal genes downregulated in Pogz−/− forebrain(A) Dot plot of DE genes in E13.5 Pogz−/− cortex and basal ganglia RNA-seq compared to wild-type controls. Significant DE genes in both tissues are indicated in red dots, n = 3. Linear regression across significant DE genes in cortex and basal ganglia, adjusted R2 = 0.77, p = 2 × 10−16.(B) Heatmap of DE genes in Pogz−/− cortex (Cx) and basal ganglia (Bg), scale is log10 of the average normalized RNA-seq reads for each gene (n = 3). Genes with a purple asterisk were validated by ISH.(C) GO analysis of Pogz−/− downregulated genes in cortex. Significant GO terms (q-value < 0.05, Benjamini-Hochberg multiple test correction) are listed.(D–L) ISH expression and validation of genes downregulated (D-K) and upregulated (L) in Pogz−/− at E13.5.
Supplier Page from Abcam for Anti-POGZ antibody