PCGF5 is required for neural differentiation of embryonic stem cells

Abstract

Polycomb repressive complex 1 (PRC1) is an important regulator of gene expression and development. PRC1 contains the E3 ligases RING1A/B, which monoubiquitinate lysine 119 at histone H2A (H2AK119ub1), and has been sub-classified into six major complexes based on the presence of a PCGF subunit. Here, we report that PCGF5, one of six PCGF paralogs, is an important requirement in the differentiation of mouse embryonic stem cells (mESCs) towards a neural cell fate. Although PCGF5 is not required for mESC self-renewal, its loss blocks mESC neural differentiation by activating the SMAD2/TGF-β signaling pathway. PCGF5 loss-of-function impairs the reduction of H2AK119ub1 and H3K27me3 around neural specific genes and keeps them repressed. Our results suggest that PCGF5 might function as both a repressor for SMAD2/TGF-β signaling pathway and a facilitator for neural differentiation. Together, our findings reveal a critical context-specific function for PCGF5 in directing PRC1 to control cell fate.

Fig. 1

PCGF5 loss-of-function blocks mESC neural differentiation. a Gene expression analysis of epigenetic factors in human embryonic stem cells (H1) and human neural stem cells (NSCs), respectively (n = 3). Results are shown relative to H1. b Schematic overview depicting the targeting strategy for the Pcgf5 locus using TALENs (PGK/PN: donor indicates that containing a loxP-flanked PGK-puromycin cassette and loxp-flanked PGK-neomycin cassette. PGK human phophoglycerol kinase promoter, P puromycin resistance gene, N neomycin resistance gene). c Western blot and qRT-PCR analysis of PCGF5 expression in wild type and Pcgf5^−/− mESCs. Results are shown relative to wild type (n = 3). d Western blot analysis of PCGF5, NANOG and OCT4 expression in wild type and Pcgf5^−/− mESCs. e Heatmap illustrating the expression of selected neurectoderm genes and mESC-specific genes that were shown as log2 FPKM in wild type and Pcgf5^−/− mESCs at day 6 after neural differentiation. Each lane corresponds to an independent biological sample. f GSEA profiles of the sets of neurectoderm genes and mESC-specific genes. g Western blot analysis of PCGF5, pluripotent markers (OCT4, NANOG), neural markers (NESTIN, β-III-TUBULIN) in wild type and Pcgf5^−/− mESCs during neural differentiation of mESCs. h–m Gene expression analysis of Nestin, Sox1, β-III-tubulin, Pcgf5, Oct4, Nanog, in wild type and Pcgf5^−/− mESCs during neural differentiation of mESCs (n = 3). Results are shown relative to wild type at day 0. Data in a, c, h–m are represented as mean values ± s.d. with the indicated significance from Student’s t-test (***p < 0.001)

Fig. 2

Loss of PCGF5 activates TGF-β signaling pathway during NPC induction. a RNA-seq-based ingenuity pathway analysis of wild type and Pcgf5^−/− mESCs at day 6 of neural differentiation. Results are showed as –log10 (p-value). b Heatmap illustrating the expression changes of the selected TGF-β-related genes that were shown as row z-score in wild type and Pcgf5^−/− mESCs at day 6 after neural differentiation. Each lane corresponds to an independent biological sample. c Gene expression analysis of the Nodal, Lefty1, Lefty2 in wild type and Pcgf5^−/− mESCs at day 6 after neural differentiation (n = 3). Results are shown relative to wild type at day 6. d Western blot analysis of pSMAD2 in wild type and Pcgf5^−/− cells at day 6 after neural differentiation. DMSO or LY2109761 (1 μM) was added during neural differentiation. e Immunostaining of the neural progenitor markers NESTIN and PAX6 in wild type and Pcgf5^−/− mESCs at day 6 after neural differentiation. Scale bar represents 100 μm. f Statistical analysis of positive cells expressing NESTIN or PAX6 described in e (n = 3). g Gene expression analysis of Pcgf5 and Sox1 at day 6 after neural differentiation. DMSO, LY2109761 (1 μM) was added into the media during neural differentiation (n = 3). Results are shown relative to wild type at day 6 after neural differentiation. h Summary of FACS data from Sox1-GFP expression in control or PCGF5-deficient mESCs at day 6 after neural differentiation. The cells were treated with DMSO, LY2109761 (1 μM), SB431542 (1 μM) or LDN-193189 HCl (100 nM) during neural differentiation (n = 3). i Statistical analysis of positive cells expressing Sox1-GFP described in h. Data in c, f, g, i, are represented as mean values ± s.d. with the indicated significance from Student’s t-test (NS, no significant, **p < 0.01, ***p < 0.001)

Fig. 3

PCGF5 stimulates RING1B-dependent histone H2AK119ub1. a Detection of the interaction between Flag-PCGF5 and H2A. Flag-tagged empty vector was used as control. b Schematic representation of Flag-PCGF5 and its deletions used in Flag co-IP assays. c Detection of the interaction between RING1B and PCGF5 with or without a Ring-finger. d Schematic representation of GST-RING1B and its deletions used in GST pull-down assays. e Proteins probed with anti-FLAG antibody by Western blot. The beads were incubated with cell lysates from the Flag-PCGF5 overexpressed in 293T cells. f Detection of the interaction between Flag-PCGF5 and PRC1 subunits by Flag co-IP in 293T cells. Flag-tagged empty vector was used as a control. g Sucrose gradient analysis of whole cell extracts of mESCs. Every fraction from a 10–30% sucrose gradient was further resolved on SDS-PAGE followed by immunoblotting for the indicated antibodies. h In vitro H2A monoubquitinylation assays with nucleosome, GST purified PCGF5, PCGF4, RING1B, E1, E2 and ubiquitin. The reactions were resolved on SDS–PAGE followed by immunoblotting. i In vitro H2A monoubquitinylation assays with purified Flag-tagged PCGF5 and Flag-tagged PCGF5 without a Ring-finger in 293T cells. The reactions were resolved on SDS-PAGE followed by immunoblotting

Fig. 4

Analysis of PCGF5 binding sites in NPCs and the PCGF5-driven deposition of H2AK119ub1 and H3K27me3 at TGF-β-associated genes during neural differentiation. a Strategy of generating Flag-tagged PCGF5 knockin stable cell lines in Sox1-GFP knockin mESCs. b Representative cellular morphologies at day 6 of neural differentiation (n = 3). The dissociated wild type and Pcgf5-3 × Flag knockin mESCs were suspended in the Petri dish and differentiated in KSR medium for 6 days. Scale bar represents 100 μm. c. Statistical analysis of Sox1-GFP cells at day 6 of neural differentiation and Western blot analysis of the Flag (Flag-PCGF5), PCGF5 in wild type and Pcgf5-3 × Flag knockin mESCs at day 6 after neural differentiation. d The tag density heatmap plot for PCGF5 binding signal. e Genome-wide distribution of PCGF5 binding sites in NPCs at day 6 after neural differentiation. f Heat map illustrating the expression changes of PCGF5 target genes that were shown as log2 fold change in wild type and Pcgf5^−/− mESCs at day 6 after neural differentiation. Each lane corresponds to an independent biological sample. g GO analysis of biological functions of upregulated and downregulated PCGF5 target genes in wild type and Pcgf5^−/− mESCs at day 6 of neural differentiation. Results are expressed as –log10 (p-value). h ChIP-qPCR analysis of PCGF5 occupancies at promoter regions of Nodal, Lefty1 and Lefty2 in both wild type and PCGF5-deficient mESCs and NPCs (at day 6 of neural differentiation) (n = 3). i ChIP-qPCR analysis of H2AK119ub1 occupancies at promoter regions of Nodal, Lefty1 and Lefty2 in both wild type and PCGF5-deficient mESCs and NPCs (at day 6 of neural differentiation) (n = 3). j ChIP-qPCR analysis of H3K27me3 occupancies at promoter regions of Nodal, Lefty1 and Lefty2 in both wild type and PCGF5-deficient mESCs and NPCs (at day 6 of neural differentiation) (n = 3). Data in c, h–j are represented as mean values ± s.d. with the indicated significance from Student’s t-test (NS no significant, *p < 0.05, ***p < 0.001)

Fig. 5

PCGF5 knockout impairs repressive chromatin from being reduced during neural differentiation. a Tag density heatmaps illustrating global changes of H2AK119ub1 in wild type and PCGF5-deficent ESCs. b Tag density heatmaps illustrating global changes of H3K27me3 in wild type and PCGF5-deficent ESCs. c Histone H2AK119ub1 occupancy (ChIP-seq read density) at three clusters of genes in both wild type and PCGF5-deficient mESCs and NPCs. d Histone H3K27me3 occupancy (ChIP-seq read density) at three clusters of genes in both wild type and PCGF5-deficient mESCs and NPCs. e GO analysis of biological functions of mESCs H2AK119ub1-enriched peaks. Results are expressed as –log10 (p-value). f GO analysis of biological functions of mESCs H3K27me3-enriched peaks. Results are expressed as –log10 (p-value). g UCSC genome browser views of binding profiles of H2AK119ub1 and H3K27me3 at the genes of Sox1, Nestin, Cdh2 and Pou3f2 in both wild type and PCGF5-deficient NPCs. h ChIP-qPCR analysis of PCGF5 occupancies at promoter regions of Sox1, Nestin, Cdh2 and Pou3f2 in both wild type and PCGF5-deficient mESCs and NPCs (at day 6 of neural differentiation) (n = 3). i ChIP-qPCR analysis of H2AK119ub1 occupancies at promoter regions of Sox1, Nestin, Cdh2 and Pou3f2 in both wild type and PCGF5-deficient mESCs and NPCs (at day 6 of neural differentiation) (n = 3). j ChIP-qPCR analysis of H3K27me3 occupancies at promoter regions of Sox1, Nestin, Cdh2 and Pou3f2 in both wild type and PCGF5-deficient mESCs and NPCs (at day 6 of neural differentiation) (n = 3). Data in h–j are represented as mean values ± s.d. with the indicated significance from Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001)