Fig 1: Zbtb20 inhibits Brn2 gene expression.(a) Quantitative RT–PCR analysis of relative mRNA abundance for the indicated proteins in Zbtb20-overexpressing and control NPCs. Data are means±s.d. (n=3). (b) E11.5 NPCs were infected with retroviruses for control, Brn2, Zbtb20 or Brn2 plus Zbtb20 and induced to differentiate for 6 days. The percentages of marker+ cells among total GFP+ cells were determined as means±s.d. (n=4). (c) Luciferase assay of relative Brn2 promoter activity and its concentration-dependent inhibition by Zbtb20 overexpression in NPCs. The effect of Neurog2 was examined as a positive control. Data are means±s.d. (n=5). (d) ChIP analysis of Zbtb20 binding to the Brn2 promoter region in NPCs. Seven different regions (R1–R7) of the Brn2 locus were tested in control cells and cells expressing TY1-tagged Zbtb20. Data are expressed as fold enrichment relative to the corresponding value for control cells and normal mouse immunoglobulin G (IgG). Data are means±s.d. (n=3 to 5). ORF, open reading frame. (e–g) Control, sh-Zbtb20 #1, sh-Zbtb20 #2 or sh-Zbtb20 #1 and mut-Zbtb20 plasmids were electroporated into P0 mouse neocortical NPCs. The P7 brain (CC, corpus callosum) was immunostained for Brn2 (e), NeuN, Tbr1 (g) and GFP. Arrows indicate marker+/GFP+ cells. The boxed region in e is shown at higher magnification in the right-most panel. Scale bars, 50 and 25 µm (higher magnification image in e). The percentages of Brn2+ cells among total GFP+ cells were determined as means±s.d. (n=5 to 7) (f). (h) Luciferase assay of relative Brn2 promoter activity in NPCs transfected with plasmids for Zbtb20, Sox9, NFIA, sh-Sox9 #1 or sh-NFIA #1, as indicated. Data are means±s.d. (n=5). *P<0.01 and **P<0.05 versus the corresponding control value (a,b,f,h), 0 µg of Zbtb20 plasmid (c) or TY1-Zbtb20, mouse IgG (d); #P<0.01 versus value for Zbtb20 (b,h) or sh-Zbtb20 #1 (f). (i) Model for astrocytogenesis by Zbtb20, Sox9 and NFIA. Zbtb20 and NFIA suppress Brn2 expression.
Fig 2: Characterization of a novel S-HSCR-associated regulatory element in intron 10 of PIK3C2B. (A) Overview of ATAC-seq and ChIP-seq profiles showing the putative hNC-specific regulatory element in PIK3C2B intron 10. The red shaded region indicates the location of the regulatory element and the line shows the A > T variant (rs551359143) found exclusively in the S-HSCR cases that disrupts the NFIA binding motif. The motif is not drawn to the same scale as the genomic signal tracks, with magnified characters. (B) Design of sgRNAs used for the CRISPR-Cas9 system for deleting the regulatory element. Genotyping reveals the specific deletion of the 171-bp fragment in intron 10 of PIK3C2B in the PIK3C2B-int10-KO hPSC line. (WT) Wild type, (KO) knockout. (C) Immunostaining shows that both the control and mutant (PIK3C2B-int1-KO) lines have comparable capability to make hNCs and hNPs. Scale bars: (hNC) 100 µm; (hNP) 200 µm. (D) RT-qPCR shows the changes in the expression of PIK3CB in different cell stages in the control and mutant lines. t-test, (ns) not significant. n = 3–4 per group. (E) Design of the constructs used for the luciferase assay. The bar chart shows the relative luciferase activities when the cells were transfected with different sets of constructs as indicated. Three independent assays were performed, each in triplicate. One-way ANOVA. (F) Gel mobility shift assays were performed with biotin-labeled probes containing the PIK3C2B intron 10 regulatory element with or without the A > T conversion and the nuclear extract from NFIA-overexpressing cells, in the presence of unlabeled probes or anti-NFIA antibody (0.1 µg). (G) Significant contacts (FDR < 0.05) in the promoter capture Hi-C data from GM12878 cells at the PIK3C2B locus. The putative regulatory element in intron 10 of PIK3C2B is marked in yellow. Contacts between the regulatory element and the TSSs of SOX13, PPP1R15B, and PIK3C2B are shown in purple curves, while contacts between the regulatory element and other promoters are shown in gray curves. Contacts that extend too far are trimmed. (H) RT-qPCR analysis shows the changes in the expression of PPP1R15B and SOX13 in the control and the mutant lines at different cell stages. t-test, (ns) not significant. n = 3–4 per group.
Fig 3: Regulation of Zbtb20 expression by gliogenic factors and effect of Zbtb20 knockdown on astrocyte differentiation induced by CNTF.(a,b) E11.5 NPCs were cultured without (No treatment) or with the indicated factors for 1 day (a) or they were infected with control, Sox9, NFIA, NICD or STAT3-C retroviruses and then cultured under differentiation-inducing conditions for 1day (b). The relative abundance of Zbtb20 mRNA was then measured by quantitative RT–PCR analysis. Data are means±s.d. (n=3). (c,d) E11.5 NPCs were infected with control, Sox9, NFIA, NICD or STAT3-C retroviruses and then cultured without FGF2 and EGF for 1 day. Control virus-infected cells were also treated with CNTF or BMP4 for 1 day. All cells were then subjected to immunostaining for Zbtb20 and GFP (c). Arrows indicate GFP+/Zbtb20+ cells. Scale bar, 50 µm. The percentages of Zbtb20+ cells among total GFP+ cells were quantified as means±s.d. (n=3) (d). (e,f) E11.5 NPCs were infected with control (sh-Luc) or Zbtb20 shRNA retroviruses, cultured without (Mock) or with CNTF for 6 days and then stained for TuJ1 (e), GFAP (f) and GFP for determination of the percentages of marker+ cells among total GFP+ cells (means±s.d., n=3). *P<0.01 versus value for No treatment (a), control virus-infected cells (d) or mock-treated cells (e,f); #P<0.01 versus value for control virus-infected cells; NS, nonsignificant; P=0.63 (CNTF) and 0.41 (BMP4) versus value for No treatment (a); P=0.50 (Sox9), 0.09 (NFIA), 0.26 (NICD) and 0.40 (STAT3-C) versus control value (b).
Fig 4: The C-terminal proline rich domain is required for the transcriptional activity of NFIA.(A) A diagram showing the domain structure of NFIA. DNA binding domain (DBD) is shown in blue, proline-rich domain is shown in pink, and transactivation domain (TAD) is shown in gray and pink combined. (B) A detailed diagram showing the C-terminal proline rich domain of murine NFIA, variant 2 as well as human NFIC, variant 1. (C) Transcriptional activities of full-length and mutant NFIA examined by luciferase reporter assay. (mean +/- S.E.M.; N = 4; ** P <0.01).
Fig 5: Direct binding of full-length NFIA to the Pparg2 77kb enhancer is indispensable for NFIA-driven adipogenesis.(A) FAIRE-seq and NFIA ChIP-seq tracks in indicated cells at day 0 of differentiation at the Pparg locus. (B) FAIRE-qPCR analysis of indicated loci (mean +/- S.E.M.; N = 3; * P <0.05, ** P <0.01). (C) ChIP-qPCR analysis for 3xFLAG-full-length NFIA and 3xFLAG-?pro#3 mutant of indicated loci using the FLAG M2 antibody. (mean +/- S.E.M.; N = 2). The representative result of multiple independent experiments is shown. (D) NFIA-expressing C2C12 myoblasts with lentivirally introduced dCas9-Krab and indicated guide RNA were stained with Oil-Red-O seven days after inducing adipocyte differentiation. (E) qPCR analysis of indicated genes in NFIA-expressing C2C12 myoblasts with lentivirally introduced dCas9-Krab and indicated guide RNA (mean +/- S.E.M.; N = 3).
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