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Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation.

Blackledge NP, Farcas AM, Kondo T, King HW, McGouran JF, Hanssen LL, Ito S, Cooper S, Kondo K, Koseki Y, Ishikura T, Long HK, Sheahan TW, Brockdorff N, Kessler BM, Koseki H, Klose RJ - Cell (2014)

Bottom Line: Chromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development.Here, using a de novo targeting assay in mouse embryonic stem cells we unexpectedly discover that PRC1-dependent H2AK119ub1 leads to recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain.This activity is restricted to variant PRC1 complexes, and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by KDM2B to CpG islands is required for normal polycomb domain formation and mouse development.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chromatin Biology and Transcription, Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, UK.

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A Model Cell System to Inducibly Disrupt Targeting of the PCGF1/PRC1 Complex, Related to Figure 5(A) A schematic of the Kdm2b gene showing the long form (Kdm2b-LF) and short form (Kdm2b-SF) transcription start sites. The positions of LoxP sites are highlighted flanking the exon which encodes the ZF-CxxC domain.(B) PCR with primers spanning the floxed exon was performed on genomic DNA from the Kdm2bfl/fl cells (UNT), the Kdm2bfl/fl cells treated for 72 hr with tamoxifen (72 hr), and wild-type cells (WT). 72hrs of tamoxifen treatment leads to a clear deletion of the floxed exon.(C) Western blot analysis indicates that loss of the KDM2B ZF-CxxC domain does not lead to destabilization of the PCGF1 and RING1B components of the KDM2B variant PRC1 complex or upregulation of its paralogue KDM2A. Furthermore, PRC2 remains present as indicated by normal levels of SUZ12.(D) Affinity purification of full-length epitope tagged wild-type (WT) KDM2B and KDM2BΔCxxC followed by tandem mass spectrometry-based analysis of associated proteins. The mascot score and percentage coverage is indicated for the KDM2B/PRC1 complex components. Importantly, removal of the ZF-CxxC exon generates a product that still associates with the PCGF1/PRC1 variant complex but lacks its capacity to bind nonmethylated DNA.
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figs5: A Model Cell System to Inducibly Disrupt Targeting of the PCGF1/PRC1 Complex, Related to Figure 5(A) A schematic of the Kdm2b gene showing the long form (Kdm2b-LF) and short form (Kdm2b-SF) transcription start sites. The positions of LoxP sites are highlighted flanking the exon which encodes the ZF-CxxC domain.(B) PCR with primers spanning the floxed exon was performed on genomic DNA from the Kdm2bfl/fl cells (UNT), the Kdm2bfl/fl cells treated for 72 hr with tamoxifen (72 hr), and wild-type cells (WT). 72hrs of tamoxifen treatment leads to a clear deletion of the floxed exon.(C) Western blot analysis indicates that loss of the KDM2B ZF-CxxC domain does not lead to destabilization of the PCGF1 and RING1B components of the KDM2B variant PRC1 complex or upregulation of its paralogue KDM2A. Furthermore, PRC2 remains present as indicated by normal levels of SUZ12.(D) Affinity purification of full-length epitope tagged wild-type (WT) KDM2B and KDM2BΔCxxC followed by tandem mass spectrometry-based analysis of associated proteins. The mascot score and percentage coverage is indicated for the KDM2B/PRC1 complex components. Importantly, removal of the ZF-CxxC exon generates a product that still associates with the PCGF1/PRC1 variant complex but lacks its capacity to bind nonmethylated DNA.

Mentions: The observation that KDM2B can nucleate PRC1 and PRC2 provided an opportunity to examine whether targeting of the PCGF1/PRC1 variant complex to natural CpG island target sites is important for polycomb domain formation. To achieve this, a novel genetic system was designed in which an exon that encodes most of the KDM2B ZF-CxxC domain and is shared by both the long and short form of the protein (Figures S4 and S5A) (Fukuda et al., 2011) was flanked by loxP sites (Kdm2bfl/fl) (Figure 5D). Homozygous Kdm2bfl/fl mouse ESC lines were then derived that also stably express a tamoxifen-inducible form of Cre-recombinase. Addition of tamoxifen rapidly yielded KDM2B long and short form proteins that lack the ZF-CxxC domain (Figures 5E and S5B), but remain associated with the PCGF1/PRC1 variant complex (Figure S5D). Importantly, cellular levels of PRC1 and PRC2 components were unaffected (Figure S5C). ChIP-seq for KDM2B in the Kdm2bfl/fl cells revealed KDM2B occupancy at CpG islands as previously described (Figure 5F) (Farcas et al., 2012; He et al., 2013; Wu et al., 2013). However, tamoxifen-mediated deletion of the ZF-CxxC domain caused a near complete loss of KDM2B chromatin occupancy and removal of PCGF1 from CpG islands (Figures 5F and 5G). Therefore, deletion of the KDM2B ZF-CxxC domain is sufficient to ablate normal targeting of the PCGF1/PRC1 complex in vivo.


Variant PRC1 complex-dependent H2A ubiquitylation drives PRC2 recruitment and polycomb domain formation.

Blackledge NP, Farcas AM, Kondo T, King HW, McGouran JF, Hanssen LL, Ito S, Cooper S, Kondo K, Koseki Y, Ishikura T, Long HK, Sheahan TW, Brockdorff N, Kessler BM, Koseki H, Klose RJ - Cell (2014)

A Model Cell System to Inducibly Disrupt Targeting of the PCGF1/PRC1 Complex, Related to Figure 5(A) A schematic of the Kdm2b gene showing the long form (Kdm2b-LF) and short form (Kdm2b-SF) transcription start sites. The positions of LoxP sites are highlighted flanking the exon which encodes the ZF-CxxC domain.(B) PCR with primers spanning the floxed exon was performed on genomic DNA from the Kdm2bfl/fl cells (UNT), the Kdm2bfl/fl cells treated for 72 hr with tamoxifen (72 hr), and wild-type cells (WT). 72hrs of tamoxifen treatment leads to a clear deletion of the floxed exon.(C) Western blot analysis indicates that loss of the KDM2B ZF-CxxC domain does not lead to destabilization of the PCGF1 and RING1B components of the KDM2B variant PRC1 complex or upregulation of its paralogue KDM2A. Furthermore, PRC2 remains present as indicated by normal levels of SUZ12.(D) Affinity purification of full-length epitope tagged wild-type (WT) KDM2B and KDM2BΔCxxC followed by tandem mass spectrometry-based analysis of associated proteins. The mascot score and percentage coverage is indicated for the KDM2B/PRC1 complex components. Importantly, removal of the ZF-CxxC exon generates a product that still associates with the PCGF1/PRC1 variant complex but lacks its capacity to bind nonmethylated DNA.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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figs5: A Model Cell System to Inducibly Disrupt Targeting of the PCGF1/PRC1 Complex, Related to Figure 5(A) A schematic of the Kdm2b gene showing the long form (Kdm2b-LF) and short form (Kdm2b-SF) transcription start sites. The positions of LoxP sites are highlighted flanking the exon which encodes the ZF-CxxC domain.(B) PCR with primers spanning the floxed exon was performed on genomic DNA from the Kdm2bfl/fl cells (UNT), the Kdm2bfl/fl cells treated for 72 hr with tamoxifen (72 hr), and wild-type cells (WT). 72hrs of tamoxifen treatment leads to a clear deletion of the floxed exon.(C) Western blot analysis indicates that loss of the KDM2B ZF-CxxC domain does not lead to destabilization of the PCGF1 and RING1B components of the KDM2B variant PRC1 complex or upregulation of its paralogue KDM2A. Furthermore, PRC2 remains present as indicated by normal levels of SUZ12.(D) Affinity purification of full-length epitope tagged wild-type (WT) KDM2B and KDM2BΔCxxC followed by tandem mass spectrometry-based analysis of associated proteins. The mascot score and percentage coverage is indicated for the KDM2B/PRC1 complex components. Importantly, removal of the ZF-CxxC exon generates a product that still associates with the PCGF1/PRC1 variant complex but lacks its capacity to bind nonmethylated DNA.
Mentions: The observation that KDM2B can nucleate PRC1 and PRC2 provided an opportunity to examine whether targeting of the PCGF1/PRC1 variant complex to natural CpG island target sites is important for polycomb domain formation. To achieve this, a novel genetic system was designed in which an exon that encodes most of the KDM2B ZF-CxxC domain and is shared by both the long and short form of the protein (Figures S4 and S5A) (Fukuda et al., 2011) was flanked by loxP sites (Kdm2bfl/fl) (Figure 5D). Homozygous Kdm2bfl/fl mouse ESC lines were then derived that also stably express a tamoxifen-inducible form of Cre-recombinase. Addition of tamoxifen rapidly yielded KDM2B long and short form proteins that lack the ZF-CxxC domain (Figures 5E and S5B), but remain associated with the PCGF1/PRC1 variant complex (Figure S5D). Importantly, cellular levels of PRC1 and PRC2 components were unaffected (Figure S5C). ChIP-seq for KDM2B in the Kdm2bfl/fl cells revealed KDM2B occupancy at CpG islands as previously described (Figure 5F) (Farcas et al., 2012; He et al., 2013; Wu et al., 2013). However, tamoxifen-mediated deletion of the ZF-CxxC domain caused a near complete loss of KDM2B chromatin occupancy and removal of PCGF1 from CpG islands (Figures 5F and 5G). Therefore, deletion of the KDM2B ZF-CxxC domain is sufficient to ablate normal targeting of the PCGF1/PRC1 complex in vivo.

Bottom Line: Chromatin modifying activities inherent to polycomb repressive complexes PRC1 and PRC2 play an essential role in gene regulation, cellular differentiation, and development.Here, using a de novo targeting assay in mouse embryonic stem cells we unexpectedly discover that PRC1-dependent H2AK119ub1 leads to recruitment of PRC2 and H3K27me3 to effectively initiate a polycomb domain.This activity is restricted to variant PRC1 complexes, and genetic ablation experiments reveal that targeting of the variant PCGF1/PRC1 complex by KDM2B to CpG islands is required for normal polycomb domain formation and mouse development.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chromatin Biology and Transcription, Department of Biochemistry, University of Oxford, Oxford, OX1 3QU, UK.

Show MeSH