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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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Failure to Target PCGF1/PRC1 Results in Homeotic Phenotypes and Embryonic Lethality(A) Kdm2bwt/ΔCxxC mice were mated with wild-type mice and at 11 day postnatal (dpn) offspring were genotyped. Results are summarized in a table.(B) A schematic summarizing the homeotic phenotypes observed in newborn Kdm2bwt/ΔCxxC mice (n = 10) with the normal wild-type vertebrae organization shown for comparison. The numbers in parentheses indicate the frequency of individual transformations.(C) Examples of the vertebral column in wild-type and Kdm2bwt/ΔCxxC mice showing homeotic phenotypes. Top: 11 dpn heterozygotes that have additional ossification at the C7 position indicating partial posteriorization (white triangles). Center: comparison of newborn wild-type and newborn Kdm2bwt/ΔCxxC mice missing dorsal processes or that have the position of the process repositioned to the anterior (black arrowhead). Bottom: 6th lumbar vertebral column transformed to sacrum in Kdm2bwt/ΔCxxC mice (red arrowhead).(D) Kdm2b homozygous  embryos recovered at 9 dpc exhibited severe developmental delay (n = 5) or in some cases only extraembryonic development (n = 2) (representative examples are shown in center and right, respectively). A heterozygote sibling is shown to indicate expected development at this stage (left).
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fig7: Failure to Target PCGF1/PRC1 Results in Homeotic Phenotypes and Embryonic Lethality(A) Kdm2bwt/ΔCxxC mice were mated with wild-type mice and at 11 day postnatal (dpn) offspring were genotyped. Results are summarized in a table.(B) A schematic summarizing the homeotic phenotypes observed in newborn Kdm2bwt/ΔCxxC mice (n = 10) with the normal wild-type vertebrae organization shown for comparison. The numbers in parentheses indicate the frequency of individual transformations.(C) Examples of the vertebral column in wild-type and Kdm2bwt/ΔCxxC mice showing homeotic phenotypes. Top: 11 dpn heterozygotes that have additional ossification at the C7 position indicating partial posteriorization (white triangles). Center: comparison of newborn wild-type and newborn Kdm2bwt/ΔCxxC mice missing dorsal processes or that have the position of the process repositioned to the anterior (black arrowhead). Bottom: 6th lumbar vertebral column transformed to sacrum in Kdm2bwt/ΔCxxC mice (red arrowhead).(D) Kdm2b homozygous embryos recovered at 9 dpc exhibited severe developmental delay (n = 5) or in some cases only extraembryonic development (n = 2) (representative examples are shown in center and right, respectively). A heterozygote sibling is shown to indicate expected development at this stage (left).

Mentions: Perturbation of Ring1a or Ring1b in mice causes axial skeletal transformations (del Mar Lorente et al., 2000; Suzuki et al., 2002) due to defects in Hox gene expression, while deletion of Ring1b alone or both Ring1a and Ring1b leads to embryonic lethality (Posfai et al., 2012; Voncken et al., 2003). To understand how PCGF1/PRC1 targeting affects development, mice hemizygous for loss of the KDM2B ZF-CxxC domain were generated by crossing Kdm2bfl/fl mice to a mouse constitutively expressing Cre-recombinase. Initial observations suggested that loss of the KDM2B ZF-CxxC domain was semi-lethal as few heterozygous mice were recovered. When Kdm2bwt/ΔCxxC were mated to wild-type mice only 20% of offspring at 10 days postnatal (dpn) were Kdm2bwt/ΔCxxC, suggesting partial haploinsufficiency (Figure 7A). To examine if the Kdm2bwt/ΔCxxC mice exhibited homeotic transformations, skeletal preparations from newborn (n = 10) and 11 dpn (n = 2) Kdm2bwt/ΔCxxC mice were compared to control Kdm2bwt/wt mice. All of the Kdm2bwt/ΔCxxC heterozygous animals exhibited skeletal alterations with homeotic transformations in cervical to sacral regions (Figures 7B and 7C). Notably, seven of the ten newborn and both of the 11 dpn Kdm2bwt/ΔCxxC mice had extra bony elements at vertebrae C7, suggesting partial transformation into T1 (Kondo and Duboule, 1999). While the second thoracic vertebra (T2) usually has a dorsal process, two of the newborn heterozygotes showed a dorsal process at T1 suggesting T1 to T2 transformation. Furthermore, dorsal processes were absent from T2 in six newborn and both 11 dpn heterozygotes, suggesting transformation of T2 to T3. Finally, two of the newborn heterozygotes showed L6-S1 transformations. Together, these homeotic phenotypes indicate posterior transformation of the vertebral column and phenocopy classical polycomb mutations (Akasaka et al., 1996; van der Lugt et al., 1994).


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)

Failure to Target PCGF1/PRC1 Results in Homeotic Phenotypes and Embryonic Lethality(A) Kdm2bwt/ΔCxxC mice were mated with wild-type mice and at 11 day postnatal (dpn) offspring were genotyped. Results are summarized in a table.(B) A schematic summarizing the homeotic phenotypes observed in newborn Kdm2bwt/ΔCxxC mice (n = 10) with the normal wild-type vertebrae organization shown for comparison. The numbers in parentheses indicate the frequency of individual transformations.(C) Examples of the vertebral column in wild-type and Kdm2bwt/ΔCxxC mice showing homeotic phenotypes. Top: 11 dpn heterozygotes that have additional ossification at the C7 position indicating partial posteriorization (white triangles). Center: comparison of newborn wild-type and newborn Kdm2bwt/ΔCxxC mice missing dorsal processes or that have the position of the process repositioned to the anterior (black arrowhead). Bottom: 6th lumbar vertebral column transformed to sacrum in Kdm2bwt/ΔCxxC mice (red arrowhead).(D) Kdm2b homozygous  embryos recovered at 9 dpc exhibited severe developmental delay (n = 5) or in some cases only extraembryonic development (n = 2) (representative examples are shown in center and right, respectively). A heterozygote sibling is shown to indicate expected development at this stage (left).
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fig7: Failure to Target PCGF1/PRC1 Results in Homeotic Phenotypes and Embryonic Lethality(A) Kdm2bwt/ΔCxxC mice were mated with wild-type mice and at 11 day postnatal (dpn) offspring were genotyped. Results are summarized in a table.(B) A schematic summarizing the homeotic phenotypes observed in newborn Kdm2bwt/ΔCxxC mice (n = 10) with the normal wild-type vertebrae organization shown for comparison. The numbers in parentheses indicate the frequency of individual transformations.(C) Examples of the vertebral column in wild-type and Kdm2bwt/ΔCxxC mice showing homeotic phenotypes. Top: 11 dpn heterozygotes that have additional ossification at the C7 position indicating partial posteriorization (white triangles). Center: comparison of newborn wild-type and newborn Kdm2bwt/ΔCxxC mice missing dorsal processes or that have the position of the process repositioned to the anterior (black arrowhead). Bottom: 6th lumbar vertebral column transformed to sacrum in Kdm2bwt/ΔCxxC mice (red arrowhead).(D) Kdm2b homozygous embryos recovered at 9 dpc exhibited severe developmental delay (n = 5) or in some cases only extraembryonic development (n = 2) (representative examples are shown in center and right, respectively). A heterozygote sibling is shown to indicate expected development at this stage (left).
Mentions: Perturbation of Ring1a or Ring1b in mice causes axial skeletal transformations (del Mar Lorente et al., 2000; Suzuki et al., 2002) due to defects in Hox gene expression, while deletion of Ring1b alone or both Ring1a and Ring1b leads to embryonic lethality (Posfai et al., 2012; Voncken et al., 2003). To understand how PCGF1/PRC1 targeting affects development, mice hemizygous for loss of the KDM2B ZF-CxxC domain were generated by crossing Kdm2bfl/fl mice to a mouse constitutively expressing Cre-recombinase. Initial observations suggested that loss of the KDM2B ZF-CxxC domain was semi-lethal as few heterozygous mice were recovered. When Kdm2bwt/ΔCxxC were mated to wild-type mice only 20% of offspring at 10 days postnatal (dpn) were Kdm2bwt/ΔCxxC, suggesting partial haploinsufficiency (Figure 7A). To examine if the Kdm2bwt/ΔCxxC mice exhibited homeotic transformations, skeletal preparations from newborn (n = 10) and 11 dpn (n = 2) Kdm2bwt/ΔCxxC mice were compared to control Kdm2bwt/wt mice. All of the Kdm2bwt/ΔCxxC heterozygous animals exhibited skeletal alterations with homeotic transformations in cervical to sacral regions (Figures 7B and 7C). Notably, seven of the ten newborn and both of the 11 dpn Kdm2bwt/ΔCxxC mice had extra bony elements at vertebrae C7, suggesting partial transformation into T1 (Kondo and Duboule, 1999). While the second thoracic vertebra (T2) usually has a dorsal process, two of the newborn heterozygotes showed a dorsal process at T1 suggesting T1 to T2 transformation. Furthermore, dorsal processes were absent from T2 in six newborn and both 11 dpn heterozygotes, suggesting transformation of T2 to T3. Finally, two of the newborn heterozygotes showed L6-S1 transformations. Together, these homeotic phenotypes indicate posterior transformation of the vertebral column and phenocopy classical polycomb mutations (Akasaka et al., 1996; van der Lugt et al., 1994).

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