Essential Role for Polycomb Group Protein Pcgf6 in Embryonic Stem Cell Maintenance and a Noncanonical Polycomb Repressive Complex 1 (PRC1) Integrity

Abstract

The Polycomb group (PcG) proteins have an important role in controlling the expression of key genes implicated in embryonic development, differentiation, and decision of cell fates. Emerging evidence suggests that Polycomb repressive complexes 1 (PRC1) is defined by the six Polycomb group RING finger protein (Pcgf) paralogs, and Pcgf proteins can assemble into noncanonical PRC1 complexes. However, little is known about the precise mechanisms of differently composed noncanonical PRC1 in the maintenance of the pluripotent cell state. Here we disrupt the Pcgf genes in mouse embryonic stem cells by CRISPR-Cas9 and find Pcgf6 null embryonic stem cells display severe defects in self-renewal and differentiation. Furthermore, Pcgf6 regulates genes mostly involved in differentiation and spermatogenesis by assembling a noncanonical PRC1 complex PRC1.6. Notably, Pcgf6 deletion causes a dramatic decrease in PRC1.6 binding to target genes and no loss of H2AK119ub1. Thus, Pcgf6 is essential for recruitment of PRC1.6 to chromatin. Our results reveal a previously uncharacterized, H2AK119ub1-independent chromatin assembly associated with PRC1.6 complex.

Keywords: Polycomb; chromatin; embryonic stem cell; epigenetics; transcription regulation.

FIGURE 1.

Generation of Pcgf6 null ES cells by two sgRNAs (spanning exon 2 to 3) guiding Cas9. A, schematic overview of the strategy to delete a genomic fragment (407 bp) containing exons 2–3 of the pcgf6 gene. PAM sequences are in red after the sgRNA sequence highlighted in blue. The scissor symbol indicates the predicted Cas9 cleavage position of the sgRNA sequence. The locations of genomic PCR primers (F, forward; R, reverse) are indicated by green arrows (external to the deletion). B, direct sequencing of the PCR product from pcgf6^−/− confirmed deletion of the 407-bp DNA fragment. Each DSB occurred exactly 3 bp upstream of the PAM sequence. C, genotyping of pcgf6 knock-out ES cells using primers upstream and downstream of the deleted region. D, RT-PCR analysis for residual Pcgf6 mRNA revealed a shorter band in the mutant consistent with a shorter message as predicted. β-Actin served as a loading control. E, Western blot analysis using an antibody recognizing pcgf6 demonstrated the absence of the protein in knock-out ES cells. Note that no mutant protein band was detected from pcgf6^−/− ES samples.

FIGURE 2.

Pcgf6 is an indispensable Pcgf family member in ES cell self-renewal. A, phase-contrast pictures of ES cells of indicated genotypes growing on feeders cells. Notably, Pcgf6^−/− ES cell colony size was strikingly reduced but fully restored by lentiviral expression of Pccgf6-F. Shown was colony-size 6 days after seeding single cell suspensions onto MEF feeder layers in the presence of LIF. B, Western blot demonstrating changes in the levels of Oct4, Sox2, and PcG proteins and transcription factors in ES cells of the indicated genotypes. β-Actin was used as a loading control. C, the maintenance of alkaline phosphatase positive colonies in Pcgf6^−/− ES cells.

FIGURE 3.

EB formation and differentiation. A, EBs were formed in hanging drops and subsequently maintained in rotating cultures. B, ES cells lacking Pcgf6 retained the potential to form EBs after LIF withdrawal, but EBs were compromised for growth. C, bar graphs indicate the mean diameters of 20 EBs from cultures of indicated genotypes. D, real-time RT-PCR was used to measure the expression of pluripotency and lineage-specific markers in control and Pcgf6^−/− mRNA samples derived from ES cells and in 12-day differentiated EBs. Marker genes for all germ layers were expressed in mutant and wild type EBs, but endodermal marker genes were significantly overexpressed at different time points in Pcgf6^−/− EBs. Shown is the mean relative mRNA expression (ratio of measured mRNA/to β-actin mRNA).

FIGURE 4.

Gene expression profiling in ES cells lacking Pcgf6. A, log₂ expression values in Pcgf6^−/− versus wild type ES cells from expression microarray. Probes were considered to be up-regulated (red) or down-regulated (green) if they had a log₂-fold change of >1 or <−1, respectively. The number of differentially expressed genes they represented is indicated. B, microarray heat map depicting gene expression in wild type, Pcgf6^−/− and Pcgf6^−/− infected with Pcgf6-F ES cells. Columns showed 2542 bars that represent genes with >2-fold expression differences between wild type (second column) and Pcgf6^−/− ES cells (third column). C, Gene Ontology analysis of Pcgf6 target genes. Shown is Gene Ontology analysis of biological functions of Pcgf6 up-regulated target genes; p values were plotted in −log; PiRNA, Piwi-interacting RNA. D, -fold changes in the expression of the top 49 genes, each of which showed >10-fold up-regulation in Pcgf6^−/− compared with wild type. Red bars, germ cell-specific genes; green bars, XLR (X-chromosome-linked lymphocyte-regulated) gene family. Microarray data were deposited at the Gene Expression Omnibus under accession number GSE92476.

FIGURE 5.

Structure/function analysis of Pcgf6 in ES cells. A, schematic representation of PCGF6 deletion and point mutants in this study and summary of their ability to rescue Pcgf6^−/− ES cell growth defect. The thick solid bars indicate the region of Pcgf6 encoded by each mutant. Deletions in the Pcgf6 coding regions are depicted as thin bent lines. The numbers representing each mutant also indicate the amino acid residue numbers at the boundaries of the deletions. B, Western blot analysis demonstrated the protein expression level of all mutants. Serial Pcgf6 deletion constructs were transfected into Pcgf6^−/− ES cells and analyzed by Western blotting with anti-FLAG antibody. Of note, a Ser³⁴→Ala³⁴ (S34A) point mutant and deletion mutants (Δ2–50, Δ2–100) of Pcgf6 were not phosphorylated. C, transcriptional ability of Pcgf6 deletion and point mutants. Changes in expression levels for the selected Pcgf6 target genes as determined by real-time PCR. Expression levels were normalized to a β-actin control and depicted as -fold changes relative to the wild type ES cells. Error bars are based on the S.D. as derived from triplicate PCR reactions.

FIGURE 6.

Essential role for Pcgf6 in PRC1. 6 complex integrity. A, physical interaction of L3mbtl2, Pcgf6, Ring1b, and Rybp in cell extracts from wild type and Pcgf6^−/− ES cells demonstrated by reciprocal co-immunoprecipitation assays. Antibodies used for immunoprecipitation (IP, top) and immunoblotting are indicated. B, the Ring domain is required for interaction of Pcgf6 with Ring1a, Ring1b, and Rybp. FLAG-Pcgf6 wild type (WT) and mutants (S34A, Δ137–175, C137A/C140A) stable-transfected into Pcgf6^−/− ES cells were immunoprecipitated with the FLAG antibody and analyzed by Western blot with the indicated specific antibodies.

FIGURE 7.

Pcgf6 colocalized with baseline levels of H2AK119ub1 at its target genes. A, expression analysis by RT-qPCR of RNA from ES cells for selected genes. Shown is relative mRNA expression (ratio of measured mRNA/to β-actin mRNA). B, ChIP of Pcgf6 followed by quantitative PCR analysis. Purified rabbit IgG was a negative control. C, ChIP of H2AK119ub1 and H3K27me3 followed by qPCR analysis of selected gene DNA in ES cells. A–C, bar graphs represent the mean of three independent biological samples and S.D. Significance: two-tailed Student's t test. D, Western blot demonstrating changes in the levels of H2AK119ub1, H3K27me3, Pcgf6, Ring1A, and Ring1B in ES cells of indicated genotypes. Histone 2A or β-actin was used as a loading control.

FIGURE 8.

Pcgf6 has a key role in regulating PRC1.6 complex recruitment to its target genes. A, ChIP of L3mbtl2, Ring1B, Rybp, and SUZ12 followed by qPCR analysis of selected gene DNA in ES cells. B, expression analysis by RT-qPCR of RNA from ES cells of the indicated genotype for selected genes. C, ChIP of Pcgf6 followed by quantitative PCR analysis in ES cells of indicated genotypes. Purified rabbit IgG was a negative control. A–C, bar graphs represent the mean of three independent biological samples and S.D. Significance: two-tailed Student's t test. D, Venn diagram illustrating the overlap of L3mbtl2-up-regulated genes with a gene set of Pcgf6-up-regulated target genes. E, model showing that the PRC1.6 complex is recruited by Pcgf6-L3mbtl2 interaction and independent of H2AK119ub1 (see “Discussion” for a detailed description).