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Jarid2 Methylation via the PRC2 Complex Regulates H3K27me3 Deposition during Cell Differentiation.

Sai S, Justin N, Teissandier A, Ancelin K, Portoso M, Caron M, Michaud A, Lombard B, da Rocha ST, Offer J, Loew D, Servant N, Wassef M, Burlina F, Gamblin SJ, Heard E, Margueron R - Mol. Cell (2015)

Bottom Line: This modification is recognized by the Eed core component of PRC2 and triggers an allosteric activation of PRC2's enzymatic activity.We show that Jarid2 methylation is important to promote PRC2 activity at a locus devoid of H3K27me3 and for the correct deposition of this mark during cell differentiation.Our results uncover a regulation loop where Jarid2 methylation fine-tunes PRC2 activity depending on the chromatin context.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, 26 Rue d'Ulm, 75005 Paris, France; INSERM U934, 26 Rue d'Ulm, 75005 Paris, France; CNRS UMR3215, 26 Rue d'Ulm, 75005 Paris, France.

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Jarid2 Methylation Occurs In Vivo(A) Left: western blot (WB) anti-Jarid2 and Jarid2-K116me2 on nuclear extract of E14 or Eed−/− ESC. Right: same in Ezh2 fl/fl-Rosa26-Cre-ERT2 before and after Tamoxifen treatment. Titrations correspond to 1× or 2× the amount of protein in controls.(B) WB and peptides competition experiment in ESC WT or Eed−/−. Nuclear extracts are probed with the indicated antibodies alone or in presence of the indicated peptides in 10-fold over antibody molar excess.(C) IF of Jarid2-K116me2 and Ezh2 on E14 and Eed−/− ESC.(D) IF of Jarid2 or Jarid-K116me2 and Ezh2 on E14 ESC.(E) IF in late mouse blastocyst for Eed and Jarid2-K116me2. Bottom panel is zoom-in of the top panel. Nuclei are stained with DAPI in all IF.(F) WB on whole extract of Drosophila S2 cell line and embryos probed with the indicated antibodies. See also Figure S2.
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fig2: Jarid2 Methylation Occurs In Vivo(A) Left: western blot (WB) anti-Jarid2 and Jarid2-K116me2 on nuclear extract of E14 or Eed−/− ESC. Right: same in Ezh2 fl/fl-Rosa26-Cre-ERT2 before and after Tamoxifen treatment. Titrations correspond to 1× or 2× the amount of protein in controls.(B) WB and peptides competition experiment in ESC WT or Eed−/−. Nuclear extracts are probed with the indicated antibodies alone or in presence of the indicated peptides in 10-fold over antibody molar excess.(C) IF of Jarid2-K116me2 and Ezh2 on E14 and Eed−/− ESC.(D) IF of Jarid2 or Jarid-K116me2 and Ezh2 on E14 ESC.(E) IF in late mouse blastocyst for Eed and Jarid2-K116me2. Bottom panel is zoom-in of the top panel. Nuclei are stained with DAPI in all IF.(F) WB on whole extract of Drosophila S2 cell line and embryos probed with the indicated antibodies. See also Figure S2.

Mentions: To test whether Jarid2 is methylated in vivo, we generated antibodies recognizing both Jarid2 di-methylated and tri-methylated at K116 (Jarid2-K116me2 and Jarid2-K116me3). We verified the specificity of these antibodies by dot blot on peptides and by western blot on in vitro methylated recombinant proteins (Figures S2A, S2B, and S2C). Western blot on nuclear extract of ESC line (E14) showed a robust signal, both for the di- and tri-methylated Jarid2-K116 antibodies, which was completely lost in ESC constitutively knocked out (KO) for Eed or conditionally KO for Ezh2 (SET domain deletion) (Figure 2A). We additionally performed competition experiment with Jarid2-K116 me2 and me3 peptides and showed that the signal for Jarid2 tri-methylation was competed out by the Jarid2-K116me3 peptide but not by Jarid2-K116me2 peptide (Figure 2B).


Jarid2 Methylation via the PRC2 Complex Regulates H3K27me3 Deposition during Cell Differentiation.

Sai S, Justin N, Teissandier A, Ancelin K, Portoso M, Caron M, Michaud A, Lombard B, da Rocha ST, Offer J, Loew D, Servant N, Wassef M, Burlina F, Gamblin SJ, Heard E, Margueron R - Mol. Cell (2015)

Jarid2 Methylation Occurs In Vivo(A) Left: western blot (WB) anti-Jarid2 and Jarid2-K116me2 on nuclear extract of E14 or Eed−/− ESC. Right: same in Ezh2 fl/fl-Rosa26-Cre-ERT2 before and after Tamoxifen treatment. Titrations correspond to 1× or 2× the amount of protein in controls.(B) WB and peptides competition experiment in ESC WT or Eed−/−. Nuclear extracts are probed with the indicated antibodies alone or in presence of the indicated peptides in 10-fold over antibody molar excess.(C) IF of Jarid2-K116me2 and Ezh2 on E14 and Eed−/− ESC.(D) IF of Jarid2 or Jarid-K116me2 and Ezh2 on E14 ESC.(E) IF in late mouse blastocyst for Eed and Jarid2-K116me2. Bottom panel is zoom-in of the top panel. Nuclei are stained with DAPI in all IF.(F) WB on whole extract of Drosophila S2 cell line and embryos probed with the indicated antibodies. See also Figure S2.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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fig2: Jarid2 Methylation Occurs In Vivo(A) Left: western blot (WB) anti-Jarid2 and Jarid2-K116me2 on nuclear extract of E14 or Eed−/− ESC. Right: same in Ezh2 fl/fl-Rosa26-Cre-ERT2 before and after Tamoxifen treatment. Titrations correspond to 1× or 2× the amount of protein in controls.(B) WB and peptides competition experiment in ESC WT or Eed−/−. Nuclear extracts are probed with the indicated antibodies alone or in presence of the indicated peptides in 10-fold over antibody molar excess.(C) IF of Jarid2-K116me2 and Ezh2 on E14 and Eed−/− ESC.(D) IF of Jarid2 or Jarid-K116me2 and Ezh2 on E14 ESC.(E) IF in late mouse blastocyst for Eed and Jarid2-K116me2. Bottom panel is zoom-in of the top panel. Nuclei are stained with DAPI in all IF.(F) WB on whole extract of Drosophila S2 cell line and embryos probed with the indicated antibodies. See also Figure S2.
Mentions: To test whether Jarid2 is methylated in vivo, we generated antibodies recognizing both Jarid2 di-methylated and tri-methylated at K116 (Jarid2-K116me2 and Jarid2-K116me3). We verified the specificity of these antibodies by dot blot on peptides and by western blot on in vitro methylated recombinant proteins (Figures S2A, S2B, and S2C). Western blot on nuclear extract of ESC line (E14) showed a robust signal, both for the di- and tri-methylated Jarid2-K116 antibodies, which was completely lost in ESC constitutively knocked out (KO) for Eed or conditionally KO for Ezh2 (SET domain deletion) (Figure 2A). We additionally performed competition experiment with Jarid2-K116 me2 and me3 peptides and showed that the signal for Jarid2 tri-methylation was competed out by the Jarid2-K116me3 peptide but not by Jarid2-K116me2 peptide (Figure 2B).

Bottom Line: This modification is recognized by the Eed core component of PRC2 and triggers an allosteric activation of PRC2's enzymatic activity.We show that Jarid2 methylation is important to promote PRC2 activity at a locus devoid of H3K27me3 and for the correct deposition of this mark during cell differentiation.Our results uncover a regulation loop where Jarid2 methylation fine-tunes PRC2 activity depending on the chromatin context.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, 26 Rue d'Ulm, 75005 Paris, France; INSERM U934, 26 Rue d'Ulm, 75005 Paris, France; CNRS UMR3215, 26 Rue d'Ulm, 75005 Paris, France.

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Related in: MedlinePlus