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Loss of Polycomb Group Protein Pcgf1 Severely Compromises Proper Differentiation of Embryonic Stem Cells

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ABSTRACT

The Polycomb repressive complex 1 (PRC1) is essential for fate decisions of embryonic stem (ES) cells. Emerging evidence suggests that six major variants of PRC1 complex, defined by the mutually exclusive presence of Pcgf subunit, regulate distinct biological processes, yet very little is known about the mechanism by which each version of PRC1 instructs and maintains cell fate. Here, we disrupted the Pcgf1, also known as Nspc1 and one of six Pcgf paralogs, in mouse ES cells by the CRISPR/Cas9 technology. We showed that although these mutant cells were viable and retained normal self-renewal, they displayed severe defects in differentiation in vitro. To gain a better understanding of the role of Pcgf1 in transcriptional control of differentiation, we analysed mRNA profiles from Pcgf1 deficient cells using RNA-seq. Interestingly, we found that Pcgf1 positively regulated expression of essential transcription factors involved in ectoderm and mesoderm differentiation, revealing an unexpected function of Pcgf1 in gene activation during ES cell lineage specification. Chromatin immunoprecipitation experiments demonstrated that Pcgf1 deletion caused a decrease in Ring1B and its associated H2AK119ub1 mark binding to target genes. Altogether, our results suggested an unexpected function of Pcgf1 in gene activation during ES cell maintenance.

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Pcgf1 works as a transcriptional activator.(A) Volcano plots represented differentially expressed genes in Pcgf1−/− compared to WT ES cells. Red colour indicated upregulated genes and green colour indicated downregulated genes if they had a log2 fold change of >1 or less than −1, respectively. The differentially expressed genes number were indicated at right. (B) A heat map of the 1929 rescuably expressed transcripts with >2-fold expression differences in WT and Pcgf1−/− ES cells. Red indicated high expression and blue indicated low expression. (C) GO analysis of biological functions of deregulated genes in Pcgf1−/− ES cells. (D) Fold changes in the expression levels of the top 36 downregulated genes, bars showed more than 24-fold change genes. The ectoderm specific genes are highlighted in red and the mesoderm specific genes are highlighted in blue.
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f4: Pcgf1 works as a transcriptional activator.(A) Volcano plots represented differentially expressed genes in Pcgf1−/− compared to WT ES cells. Red colour indicated upregulated genes and green colour indicated downregulated genes if they had a log2 fold change of >1 or less than −1, respectively. The differentially expressed genes number were indicated at right. (B) A heat map of the 1929 rescuably expressed transcripts with >2-fold expression differences in WT and Pcgf1−/− ES cells. Red indicated high expression and blue indicated low expression. (C) GO analysis of biological functions of deregulated genes in Pcgf1−/− ES cells. (D) Fold changes in the expression levels of the top 36 downregulated genes, bars showed more than 24-fold change genes. The ectoderm specific genes are highlighted in red and the mesoderm specific genes are highlighted in blue.

Mentions: To understand the underlying mechanism by which Pcgf1 affects ES cell differentiation, it is critical to characterize Pcgf1 regulated transcripts. To this end, we performed RNA-seq analysis on Pcgf1−/−and wild-type ES cells. RNA-seq analysis identified 2331 genes with >2-fold altered expression levels in Pcgf1−/− compared to wild-type ES cells (Fig. 4A, Supplementary Table 2). Importantly, re-expression of Pcgf1-FLAG was accompanied by 82% of these genes altered >2-fold in the opposite direction (Pcgf1-FLAG infected cells compared with Pcgf1−/−). Together, these criteria revealed a set of 1929 Pcgf1 target genes. 1491 (77%) genes were downregulated in the absence of Pcgf1 while only 438 genes (23%) were upregulated (Fig. 4B). Expression of some of the transcripts identified as downregulated by RNA-seq analysis was evaluated independently by RT-quantitative PCR (RT-qPCR) (see figure below). The RNA-seq data have been deposited at the Gene Expression Omnibus under accession number GSE95383. Thus, Pcgf1 generally functioned as a transcriptional activator in ES cells. Next, we used gene ontology (GO) analysis to identify the functions of the significantly downregulated genes. These genes were enriched in many functional categories which conformed to the differentiation phenotype we observed, like the development of mesoderm (muscle contraction, blood circulation) and ectoderm (regulation of neurotransmitter levels and synaptic signaling) (Fig. 4C). Figure 4D showed 36 genes downregulated with >24-fold decrease. As expected, these genes were mainly associated with mesoderm and ectoderm differentiation or related to pathways essential for these two germ layer differentiation (e.g. Pla2g4f, Col6a5, Col1a2, Rnls and Chrnd for mesoderm; Pclo, Ryr3, Pde6b, Calb2, Atp2b2 and Kcnj2 for ectoderm). Thus, Pcgf1 acts predominantly as a transcriptional activator which regulates mesoderm and ectoderm differentiation in ES cells.


Loss of Polycomb Group Protein Pcgf1 Severely Compromises Proper Differentiation of Embryonic Stem Cells
Pcgf1 works as a transcriptional activator.(A) Volcano plots represented differentially expressed genes in Pcgf1−/− compared to WT ES cells. Red colour indicated upregulated genes and green colour indicated downregulated genes if they had a log2 fold change of >1 or less than −1, respectively. The differentially expressed genes number were indicated at right. (B) A heat map of the 1929 rescuably expressed transcripts with >2-fold expression differences in WT and Pcgf1−/− ES cells. Red indicated high expression and blue indicated low expression. (C) GO analysis of biological functions of deregulated genes in Pcgf1−/− ES cells. (D) Fold changes in the expression levels of the top 36 downregulated genes, bars showed more than 24-fold change genes. The ectoderm specific genes are highlighted in red and the mesoderm specific genes are highlighted in blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f4: Pcgf1 works as a transcriptional activator.(A) Volcano plots represented differentially expressed genes in Pcgf1−/− compared to WT ES cells. Red colour indicated upregulated genes and green colour indicated downregulated genes if they had a log2 fold change of >1 or less than −1, respectively. The differentially expressed genes number were indicated at right. (B) A heat map of the 1929 rescuably expressed transcripts with >2-fold expression differences in WT and Pcgf1−/− ES cells. Red indicated high expression and blue indicated low expression. (C) GO analysis of biological functions of deregulated genes in Pcgf1−/− ES cells. (D) Fold changes in the expression levels of the top 36 downregulated genes, bars showed more than 24-fold change genes. The ectoderm specific genes are highlighted in red and the mesoderm specific genes are highlighted in blue.
Mentions: To understand the underlying mechanism by which Pcgf1 affects ES cell differentiation, it is critical to characterize Pcgf1 regulated transcripts. To this end, we performed RNA-seq analysis on Pcgf1−/−and wild-type ES cells. RNA-seq analysis identified 2331 genes with >2-fold altered expression levels in Pcgf1−/− compared to wild-type ES cells (Fig. 4A, Supplementary Table 2). Importantly, re-expression of Pcgf1-FLAG was accompanied by 82% of these genes altered >2-fold in the opposite direction (Pcgf1-FLAG infected cells compared with Pcgf1−/−). Together, these criteria revealed a set of 1929 Pcgf1 target genes. 1491 (77%) genes were downregulated in the absence of Pcgf1 while only 438 genes (23%) were upregulated (Fig. 4B). Expression of some of the transcripts identified as downregulated by RNA-seq analysis was evaluated independently by RT-quantitative PCR (RT-qPCR) (see figure below). The RNA-seq data have been deposited at the Gene Expression Omnibus under accession number GSE95383. Thus, Pcgf1 generally functioned as a transcriptional activator in ES cells. Next, we used gene ontology (GO) analysis to identify the functions of the significantly downregulated genes. These genes were enriched in many functional categories which conformed to the differentiation phenotype we observed, like the development of mesoderm (muscle contraction, blood circulation) and ectoderm (regulation of neurotransmitter levels and synaptic signaling) (Fig. 4C). Figure 4D showed 36 genes downregulated with >24-fold decrease. As expected, these genes were mainly associated with mesoderm and ectoderm differentiation or related to pathways essential for these two germ layer differentiation (e.g. Pla2g4f, Col6a5, Col1a2, Rnls and Chrnd for mesoderm; Pclo, Ryr3, Pde6b, Calb2, Atp2b2 and Kcnj2 for ectoderm). Thus, Pcgf1 acts predominantly as a transcriptional activator which regulates mesoderm and ectoderm differentiation in ES cells.

View Article: PubMed Central - PubMed

ABSTRACT

The Polycomb repressive complex 1 (PRC1) is essential for fate decisions of embryonic stem (ES) cells. Emerging evidence suggests that six major variants of PRC1 complex, defined by the mutually exclusive presence of Pcgf subunit, regulate distinct biological processes, yet very little is known about the mechanism by which each version of PRC1 instructs and maintains cell fate. Here, we disrupted the Pcgf1, also known as Nspc1 and one of six Pcgf paralogs, in mouse ES cells by the CRISPR/Cas9 technology. We showed that although these mutant cells were viable and retained normal self-renewal, they displayed severe defects in differentiation in vitro. To gain a better understanding of the role of Pcgf1 in transcriptional control of differentiation, we analysed mRNA profiles from Pcgf1 deficient cells using RNA-seq. Interestingly, we found that Pcgf1 positively regulated expression of essential transcription factors involved in ectoderm and mesoderm differentiation, revealing an unexpected function of Pcgf1 in gene activation during ES cell lineage specification. Chromatin immunoprecipitation experiments demonstrated that Pcgf1 deletion caused a decrease in Ring1B and its associated H2AK119ub1 mark binding to target genes. Altogether, our results suggested an unexpected function of Pcgf1 in gene activation during ES cell maintenance.

No MeSH data available.


Related in: MedlinePlus