Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Nov 1;8(1):1235.
doi: 10.1038/s41467-017-01464-w.

The Polycomb group protein CBX6 is an essential regulator of embryonic stem cell identity

Alexandra Santanach  1   2 , Enrique Blanco  1   2 , Hua Jiang  3 , Kelly R Molloy  4 , Miriam Sansó  1   2 , John LaCava  3   5 , Lluis Morey  1   2   6 , Luciano Di Croce  7   8   9
Affiliations

The Polycomb group protein CBX6 is an essential regulator of embryonic stem cell identity

Alexandra Santanach et al. Nat Commun. .

Abstract

Polycomb group proteins (PcG) are transcriptional repressors that control cell identity and development. In mammals, five different CBX proteins associate with the core Polycomb repressive complex 1 (PRC1). In mouse embryonic stem cells (ESCs), CBX6 and CBX7 are the most highly expressed CBX family members. CBX7 has been recently characterized, but little is known regarding the function of CBX6. Here, we show that CBX6 is essential for ESC identity. Its depletion destabilizes the pluripotency network and triggers differentiation. Mechanistically, we find that CBX6 is physically and functionally associated to both canonical PRC1 (cPRC1) and non-canonical PRC1 (ncPRC1) complexes. Notably, in contrast to CBX7, CBX6 is recruited to chromatin independently of H3K27me3. Taken together, our findings reveal that CBX6 is an essential component of ESC biology that contributes to the structural and functional complexity of the PRC1 complex.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
CBX6-depleted ESCs spontaneously differentiate. a Phase contrast image of shCtl, shCbx6, and shCbx7 ESC lines. b Accumulative proliferation measurements of shCtl and shCbx6 along passages. SEM, standard error of mean; P, passage; Signif., significance. Significance was analyzed through Student’s t test. Significance was considered when P value was ≤0,05. P value of ** is ≤10−2, P value of *** is ≤10−3. c RT-qPCR analysis of control and CBX6-depleted ESCs. Results are shown relative to shCtl and are normalized to the housekeeping gene Rpo. Error bars represent standard deviation (SD) of three independent experiments. Significance was analyzed through Student’s t test. Significance was considered when P value was ≤0.05 (*). d Phase contrast image of AP staining performed on shCtl or shCbx6 ESCs (right panel); quantification of the AP staining assays, representing the mean of three independent experiments in which around 40 random colonies were counted (left panel)
Fig. 2
Fig. 2
CBX6 function depends on its N- and C-terminal domains. a Schematic representation of the CBX6 constructs used in the rescue experiment. Note that every construct contained a silent mutation (not depicted) that conferred resistance to the CBX6 shRNA. b Phase contrast images of different cell lines overexpressing an empty construct or a CBX6WT, CBX6AA, or CBX6ΔPcR construct, additionally infected with shCtl or shCbx6 lentiviral particles. c qRT-PCR analysis of pluripotency genes in the different cell lines. Results are shown relative to empty shCtl and were normalized to Rpo. Error bars represent SD of two independent experiments. d qRT-PCR analysis of pluripotency genes in the shCtl-infected cell lines. Results are shown relative to empty shCtl and normalized to Rpo. Error bars represent SD of two independent experiments
Fig. 3
Fig. 3
CBX6 interactome. a Protein complex affinity capture workflow. b Statistically enriched proteins in the 3×FLAG IP identified by permutation-based FDR-corrected t-test. The plot shows log2 (difference) ratios of averaged protein intensities of the 3×FLAG pull-down over the control, plotted against the –log10 (P value). The hyperbolic significance curve was calculated based on a combination of P value and fold-change. The proteins in the upper right corner represent the bait and its interactors (marked in red). A fold-change of ≥2 and a false discovery rate (FDR) of 0.05 were considered significant. c Co-IPs from total ESC extracts using an antibody against 3×FLAG. Western blots of different proteins are shown. d Co-IPs from total CBX6-3×FLAG-expressing ESC extracts using an antibody against RING1B. Western blots of different proteins are shown
Fig. 4
Fig. 4
CBX6 genome-wide distribution features. a TSS (±5 kb) enrichment plot of CBX6 ChIP-seq at 2730 CBX6 target sites. b ChIP-qPCR validation of target genes of CBX6 in control and CBX6-3×HA-expressing cells. Results are shown relative to the input percentage. Error bars represent SD of three biological replicates. Significance was analyzed through Student’s t test. Significance was considered when P value was ≤0.05 (*). c Box plots showing expression of CBX6, H3K27me3, and H3K36me3 target genes. d GO analysis of biological functions and signaling pathways of CBX6 target genes. P values are plotted in −log. e RNAseq heat map of up- and downregulated genes in CBX6-depleted cells as compared to control cells. Only genes up- or downregulated by at least 1.5-fold as compared to control cells are shown. f Venn diagrams showing the overlap between CBX6 and CBX7 deregulated genes upon their depletion
Fig. 5
Fig. 5
CBX6 occupies cPRC1 sites. a TSS (±5 kb) enrichment plot of CBX6, RING1B, CBX7, H2AK119ub, and SUZ12 ChIP-seq at 2730 CBX6 target sites. b Venn diagrams showing the overlap of CBX6 target genes with those of RING1B CBX7, H2AK119ub, SUZ12, and PCGF6. c (left) TSS (±5 kb) enrichment plot of RING1B in shCtl and shCbx6 ESCs, at 2730 CBX6 target sites. (right) ChIP-qPCR in shCTL or shCBX6 ESCs of RING1B and H2AK119Ub. d (left) TSS (±5 kb) enrichment plot of CBX6 in shCtl and shCbx7 ESCs, at 2730 CBX6 target sites. (right) ChIP-qPCR in shCTL and shCBX6 (or shCbx7) ESCs of CBX7 (or CBX6), respectively. e ChIP-qPCR in shCTL or shCBX6 ESCs of Suz12 and H3K27me3. f ChIP-qPCR of CBX6 in shCTL and shSuz12 ESCs. c–f For all the experiments, an intergenic region was used as a negative control gene. Results are shown relative to percentage of input. For all the experiments an intergenic region was used as a negative control gene. Results are shown relative to percentage of input. Error bars represent SD of three biological replicates. Error bars represent SD of three biological replicates
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Lewis EB. A gene complex controlling segmentation in Drosophila. Nature. 1978;276:565–570. doi: 10.1038/276565a0. - DOI - PubMed
    1. Laugesen A, Helin K. Chromatin repressive complexes in stem cells, development, and cancer. Cell Stem Cell. 2014;14:735–751. doi: 10.1016/j.stem.2014.05.006. - DOI - PubMed
    1. Di Croce L, Helin K. Transcriptional regulation by Polycomb group proteins. Nat. Struct. Mol. Biol. 2013;20:1147–1155. doi: 10.1038/nsmb.2669. - DOI - PubMed
    1. Simon JA, Kingston RE. Mechanisms of polycomb gene silencing: knowns and unknowns. Nat. Rev. Mol. Cell Biol. 2009;10:697–708. doi: 10.1038/nrn2731. - DOI - PubMed
    1. Cao R, Zhang Y. The functions of E(Z)/EZH2-mediated methylation of lysine 27 in histone H3. Curr. Opin. Genet. Dev. 2004;14:155–164. doi: 10.1016/j.gde.2004.02.001. - DOI - PubMed

Publication types

Substances

LinkOut - more resources

-