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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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Both the Short and Long Form of KDM2B Mediate Polycomb Domain Formation in a Histone Demethylase Activity-Independent Manner, Related to Figure 5(A) KDM2B long form (LF) and short form (SF) with their domain organization indicated. Additional, TetR fusion constructs which have had domains removed (gray boxes) or mutated are indicated.(B) Western blot analysis of the TetR-KDM2B fusion cell lines indicating roughly equal protein expression.(C) ChIP-qPCR analysis for the TetR fusion protein, RING1B, H2AK119ub1, SUZ12, EZH2, and H3K27me3 across the TetO containing region in the TetR only, TetR-KDM2B LF, TetR-KDM2B SF, and TetR-KDM2B LF demethylase mutant (JmjC mutant). All three versions of KDM2B lead to efficient RING1B recruitment, H2AK119ub1, and formation of a polycomb domain containing SUZ12, EZH2, and H3K27me3. This indicates that both forms of KDM2B can form polycomb domains independent of their demethylase activity.(D) An epitope tagged version of the KDM2B-SF was stably expressed in mouse ESCs, affinity purified, and associated proteins identified by tandem mass spectrometry. This revealed that the short form of KDM2B forms the same variant PRC1 complex as the long form of the protein, consistent with its capacity to recruit RING1B and form polycomb domains in tethering assays.(E) Based on the capacity of KDM2B-SF to associate with the PCGF1/PRC1 complex (D) the domain(s) mediating this were further mapped in tethering assays. The C-terminal Fbox and LRR domains are required for RING1B recruitment whereas the PHD domain is dispensable.
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figs4: Both the Short and Long Form of KDM2B Mediate Polycomb Domain Formation in a Histone Demethylase Activity-Independent Manner, Related to Figure 5(A) KDM2B long form (LF) and short form (SF) with their domain organization indicated. Additional, TetR fusion constructs which have had domains removed (gray boxes) or mutated are indicated.(B) Western blot analysis of the TetR-KDM2B fusion cell lines indicating roughly equal protein expression.(C) ChIP-qPCR analysis for the TetR fusion protein, RING1B, H2AK119ub1, SUZ12, EZH2, and H3K27me3 across the TetO containing region in the TetR only, TetR-KDM2B LF, TetR-KDM2B SF, and TetR-KDM2B LF demethylase mutant (JmjC mutant). All three versions of KDM2B lead to efficient RING1B recruitment, H2AK119ub1, and formation of a polycomb domain containing SUZ12, EZH2, and H3K27me3. This indicates that both forms of KDM2B can form polycomb domains independent of their demethylase activity.(D) An epitope tagged version of the KDM2B-SF was stably expressed in mouse ESCs, affinity purified, and associated proteins identified by tandem mass spectrometry. This revealed that the short form of KDM2B forms the same variant PRC1 complex as the long form of the protein, consistent with its capacity to recruit RING1B and form polycomb domains in tethering assays.(E) Based on the capacity of KDM2B-SF to associate with the PCGF1/PRC1 complex (D) the domain(s) mediating this were further mapped in tethering assays. The C-terminal Fbox and LRR domains are required for RING1B recruitment whereas the PHD domain is dispensable.

Mentions: Deletion of RING1A/B in mouse ESCs supports a model whereby H2AK119ub1 contributes to the occupancy of PRC2 at natural target sites in vivo. However, removal of RING1A/B disrupts both canonical and variant PRC1 complex activity. Understanding if variant PRC1 complexes can drive this process at natural target sites is challenging, as variant PRC1 complex targeting mechanisms remain poorly defined. An exception is the PCGF1/PRC1 complex which contains a histone lysine demethylase protein, KDM2B, which binds to nonmethylated DNA via a ZF-CxxC DNA-binding domain (Farcas et al., 2012; He et al., 2013; Long et al., 2013; Wu et al., 2013). Nonmethylated DNA is generally concentrated in vertebrate regulatory elements called CpG islands, and most mammalian polycomb target sites are associated with CpG islands (Ku et al., 2008). KDM2B may therefore represent a direct molecular link between recognition of CpG island target sites and occupancy of both PRC1 and PRC2. To determine if KDM2B binding is sufficient to recruit the PCGF1/PRC1 complex and establish a polycomb domain de novo, a TetR-KDM2B fusion protein was stably expressed in the TetO cell line (Figure 5A). TetR-KDM2B led to RING1B, PCGF1, and H2AK119ub1 deposition (Figure 5A). This was not observed with the related KDM2A protein which does not interact with PRC1 (Figure 5A) (Blackledge et al., 2010). Importantly, PCGF1/PRC1 recruitment by KDM2B resulted in binding of PRC2 and H3K27me3 (Figures 5A and S4C). This activity was dependent on recruitment of PCGF1/PRC1, as depletion of PCGF1 in the TetR-KDM2B line caused a clear reduction in both PRC1 and PRC2 (Figures 5B and 5C). Interestingly, polycomb domain formation did not rely on KDM2B demethylase activity, as a catalytic mutant of KDM2B or a short form of the protein that lacks the demethylase domain recruited PRC1 and PRC2 to similar levels (Figures S4A–S4C). Therefore, de novo targeting of the PCGF1/PRC1 complex by KDM2B leads to polycomb domain formation in a manner similar to TetR-PCGF1 (Figure 1).


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)

Both the Short and Long Form of KDM2B Mediate Polycomb Domain Formation in a Histone Demethylase Activity-Independent Manner, Related to Figure 5(A) KDM2B long form (LF) and short form (SF) with their domain organization indicated. Additional, TetR fusion constructs which have had domains removed (gray boxes) or mutated are indicated.(B) Western blot analysis of the TetR-KDM2B fusion cell lines indicating roughly equal protein expression.(C) ChIP-qPCR analysis for the TetR fusion protein, RING1B, H2AK119ub1, SUZ12, EZH2, and H3K27me3 across the TetO containing region in the TetR only, TetR-KDM2B LF, TetR-KDM2B SF, and TetR-KDM2B LF demethylase mutant (JmjC mutant). All three versions of KDM2B lead to efficient RING1B recruitment, H2AK119ub1, and formation of a polycomb domain containing SUZ12, EZH2, and H3K27me3. This indicates that both forms of KDM2B can form polycomb domains independent of their demethylase activity.(D) An epitope tagged version of the KDM2B-SF was stably expressed in mouse ESCs, affinity purified, and associated proteins identified by tandem mass spectrometry. This revealed that the short form of KDM2B forms the same variant PRC1 complex as the long form of the protein, consistent with its capacity to recruit RING1B and form polycomb domains in tethering assays.(E) Based on the capacity of KDM2B-SF to associate with the PCGF1/PRC1 complex (D) the domain(s) mediating this were further mapped in tethering assays. The C-terminal Fbox and LRR domains are required for RING1B recruitment whereas the PHD domain is dispensable.
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figs4: Both the Short and Long Form of KDM2B Mediate Polycomb Domain Formation in a Histone Demethylase Activity-Independent Manner, Related to Figure 5(A) KDM2B long form (LF) and short form (SF) with their domain organization indicated. Additional, TetR fusion constructs which have had domains removed (gray boxes) or mutated are indicated.(B) Western blot analysis of the TetR-KDM2B fusion cell lines indicating roughly equal protein expression.(C) ChIP-qPCR analysis for the TetR fusion protein, RING1B, H2AK119ub1, SUZ12, EZH2, and H3K27me3 across the TetO containing region in the TetR only, TetR-KDM2B LF, TetR-KDM2B SF, and TetR-KDM2B LF demethylase mutant (JmjC mutant). All three versions of KDM2B lead to efficient RING1B recruitment, H2AK119ub1, and formation of a polycomb domain containing SUZ12, EZH2, and H3K27me3. This indicates that both forms of KDM2B can form polycomb domains independent of their demethylase activity.(D) An epitope tagged version of the KDM2B-SF was stably expressed in mouse ESCs, affinity purified, and associated proteins identified by tandem mass spectrometry. This revealed that the short form of KDM2B forms the same variant PRC1 complex as the long form of the protein, consistent with its capacity to recruit RING1B and form polycomb domains in tethering assays.(E) Based on the capacity of KDM2B-SF to associate with the PCGF1/PRC1 complex (D) the domain(s) mediating this were further mapped in tethering assays. The C-terminal Fbox and LRR domains are required for RING1B recruitment whereas the PHD domain is dispensable.
Mentions: Deletion of RING1A/B in mouse ESCs supports a model whereby H2AK119ub1 contributes to the occupancy of PRC2 at natural target sites in vivo. However, removal of RING1A/B disrupts both canonical and variant PRC1 complex activity. Understanding if variant PRC1 complexes can drive this process at natural target sites is challenging, as variant PRC1 complex targeting mechanisms remain poorly defined. An exception is the PCGF1/PRC1 complex which contains a histone lysine demethylase protein, KDM2B, which binds to nonmethylated DNA via a ZF-CxxC DNA-binding domain (Farcas et al., 2012; He et al., 2013; Long et al., 2013; Wu et al., 2013). Nonmethylated DNA is generally concentrated in vertebrate regulatory elements called CpG islands, and most mammalian polycomb target sites are associated with CpG islands (Ku et al., 2008). KDM2B may therefore represent a direct molecular link between recognition of CpG island target sites and occupancy of both PRC1 and PRC2. To determine if KDM2B binding is sufficient to recruit the PCGF1/PRC1 complex and establish a polycomb domain de novo, a TetR-KDM2B fusion protein was stably expressed in the TetO cell line (Figure 5A). TetR-KDM2B led to RING1B, PCGF1, and H2AK119ub1 deposition (Figure 5A). This was not observed with the related KDM2A protein which does not interact with PRC1 (Figure 5A) (Blackledge et al., 2010). Importantly, PCGF1/PRC1 recruitment by KDM2B resulted in binding of PRC2 and H3K27me3 (Figures 5A and S4C). This activity was dependent on recruitment of PCGF1/PRC1, as depletion of PCGF1 in the TetR-KDM2B line caused a clear reduction in both PRC1 and PRC2 (Figures 5B and 5C). Interestingly, polycomb domain formation did not rely on KDM2B demethylase activity, as a catalytic mutant of KDM2B or a short form of the protein that lacks the demethylase domain recruited PRC1 and PRC2 to similar levels (Figures S4A–S4C). Therefore, de novo targeting of the PCGF1/PRC1 complex by KDM2B leads to polycomb domain formation in a manner similar to TetR-PCGF1 (Figure 1).

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
Related in: MedlinePlus