Additional file 1 of Autism-associated protein POGZ controls ESCs and ESC neural induction by association with esBAF
Creators: name: Sun, Xiaoyun
name: Cheng, Linxi
name: Sun, Yuhua
Publisher: name: figshare
Year: 2022
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Figure S1 Related to Figure 1. A Genotyping showing two additional mutant alleles: Mut2 and 3. B PH3 staining of control ESCs, Mut1- and Mut2-Pogz−/− ESCs at passage 12, showing reduced number of PH3 positive cells in homologous and heterozygous mutant ESCs. CQuantification of (B). D Apoptosis analysis by flow cytometry for control-, Mut1- (passage 12), Mut2- (passage 12), and FLAG-POGZ restoring ESCs. E The qRT-PCR results for the indicated genes of control, Mut1, and Mut2 ESCs at passage 18. F IF staining results showing that GATA6 was abnormally expressed in Mut2 and Mut3 Pogz−/− ESCs. Bar: 25 μm. Figure S2 Related to Figure 2. A Morphology of day 1, 2, 6, and 8 EBs from control and two additional Pogz mutant ESCs (Mut2 and 3). B KEGG analysis of DEGs. CMorphology images during the formation of neurospheres and neuronal cells from control and Pogz−/− ESCs. Bar: 25 μm. Figure S3 Related to Figure 3. A Double IF staining of HP1γ and FLAG-POGZ. Bar: 25 μm. B IP results showing that POGZ failed to pull down CHD4 in ESCs. C IP results showing that ADNP failed to pull down POGZ in ESCs. D IP results showing that POGZ failed to pull down ADNP in ESCs. E IP results showing FLAG-POGZ-Nter failed to pull down HA-BRG1-Nter in 293T cells. F IP results showing FLAG-POGZ-Cter failed to pull down HA-BRG1-Nter in 293T cells. G IP results showing FLAG-POGZ-Nter failed to pull down HA-BRG1-Cter in 293T cells. H IP results showing HA-BRG1-Cter failed to pull down FLAG-POGZ-Nter in 293T cells. I IP of in vitro synthesized POGZ and FLAG-BAF155 by TnT system. IP: POGZ, WB: FLAG. All experiments were repeated at least two times, and shown are representative images. Figure S4 Related to Figure 4. A Snapshots of ChIP and CUT&amp;Tag signals at Chchd1 and Dcp1a loci, showing that CUT&amp;Tag outperforms FLAG ChIP-seq. Gray: proximal TSS; Orange: gene distal or gene body regions. B Snapshots ChIP and CUT&amp;Tag peaks at the indicated loci, showing that POGZ is co-localized with H3K4me1 and H3K27ac. Gray: proximal TSS; Orange: enhancer regions. C A rank of motifs identified in POGZ binding sites by HOMER. Shown were the top 18 motifs with p value. D Snapshots of ChIP-seq and CUT&amp;Tag peaks at Nanog and Pou5f1 loci, showing a broad overlapping occupancy of POGZ, OCT4, NANOG, H3K27Ac, ESRRB, KLF4, and MED1, indicating that POGZ is localized to super-enhancers. Orange: enhancer regions. Figure S5 Related to Figure 5. AGenome-wide distribution patterns of POGZ and BRG1. B Snapshots showing the overlapping of POGZ, BRG1, BAF155, and SS18 at the indicated genes. C (Up) GO terms of BRG1+POGZ- genes; (Bottom) KEGG terms of BRG1+POGZ- genes. D (Up) GO terms of BRG1-POGZ+ genes; (Bottom) KEGG terms of BRG1-POGZ+ genes. E ChIP-PCR results showing BRG1 enrichment at promoters of the indicated genes in day 5 neurospheres from control and Pogz−/− ESCs. Figure S6 A Heat map showing ATAC-seq signals in control and Pogz−/− ESCs. B Snapshots showing POGZ was enriched at ATAC-seq hypersensitive sites. C Top three motifs identified at POGZ-dependent ATAC-seq sites. D Metaplot showing that there was no change of chromatin accessibility at proximal TSS. E GO analysis of 148 genes that exhibited altered chromatin accessibility. F Snapshots showing that chromatin signals at proximal TSS and distal regions of the indicated genes. Gray: proximal TSS; Orange: distal regions. G Snapshots showing the ATAC-seq peaks at the indicated genes in the presence and absence of POGZ and BRG1. Gray highlighting the POGZ-bound DA peaks at the indicated loci. Figure S7 Related to Figure 7. A Heat map showing H3K4me3 ChIP signals in control and Pogz−/− ESCs. BHeat map showing H3K4me3 signals around the proximal TSS in control and Pogz−/− ESCs. C Snapshots showing the ChIP-seq peaks of H3K4me3 and H3K27ac at the indicated genes in control and Pogz−/− ESCs. Neural and imprinted genes in black dashed box, and pluripotency genes in red dashed box. D Snapshots showing H3K27ac ChIP-seq peaks at the indicated genes in control and Pogz−/− ESC-derived neurospheres. Orange: enhancers; Gray: promoters