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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 Is Required to Set Up Differentiation Programs in ESC(A) IF for Jarid2 and Oct4 in Jarid2 KO and Jarid2 WT or K116A rescued ESC.(B) WB with the indicated antibodies on nuclear extracts of the three ESC lines.(C) Left: colony morphology quantification of the indicated ESC. Values represent the percentage of alkaline-phosphatase-positive colonies showing a compact, flat, or spread morphology (mean ± SD, n ≥ 2). Right: number of methylene-blue-stained colonies (mean ± SD, n ≥ 2) detected 8 days after plating 100 cells per well.(D) Top: cell growth curve of the ESC over 4 days (mean ± SD, n = 3). Bottom: representative view of phosphatase alkaline staining.(E) WB probing nuclear extracts during ESC differentiation.(F) RNA transcript quantification through multiplexed-digital hybridization-based analysis (mean, n = 2). Heatmap representing the Log2-transformed median centered values. Top: messenger RNA classified by function. Bottom: transcripts from repeated sequences. Values are normalized around a set of housekeeping genes (ActB, Hprt1, Gapdh, Ppia, RplPO, and Rrm2). See also Figure S6.
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fig6: Jarid2 Methylation Is Required to Set Up Differentiation Programs in ESC(A) IF for Jarid2 and Oct4 in Jarid2 KO and Jarid2 WT or K116A rescued ESC.(B) WB with the indicated antibodies on nuclear extracts of the three ESC lines.(C) Left: colony morphology quantification of the indicated ESC. Values represent the percentage of alkaline-phosphatase-positive colonies showing a compact, flat, or spread morphology (mean ± SD, n ≥ 2). Right: number of methylene-blue-stained colonies (mean ± SD, n ≥ 2) detected 8 days after plating 100 cells per well.(D) Top: cell growth curve of the ESC over 4 days (mean ± SD, n = 3). Bottom: representative view of phosphatase alkaline staining.(E) WB probing nuclear extracts during ESC differentiation.(F) RNA transcript quantification through multiplexed-digital hybridization-based analysis (mean, n = 2). Heatmap representing the Log2-transformed median centered values. Top: messenger RNA classified by function. Bottom: transcripts from repeated sequences. Values are normalized around a set of housekeeping genes (ActB, Hprt1, Gapdh, Ppia, RplPO, and Rrm2). See also Figure S6.

Mentions: The results presented so far demonstrate that Jarid2 methylation is important for PRC2 activity in vitro and when artificially tethered to chromatin in a cellular context. To confirm the biological relevance of this regulation, we evaluated the function of Jarid2 methylation in ESC. We used ESC KO for Jarid2 (Shen et al., 2009) and generated cell lines rescued with either WT or K116A mutant forms of Jarid2. Since Jarid2 is dynamically regulated during cell differentiation, the rescues were performed using bacterial artificial chromosomes (BACs) in order to recapitulate the endogenous expression of the Jarid2 gene. Rescued ESC lines expressed Jarid2 homogeneously, with appropriate nuclear distributions as illustrated by IF (Figure 6A) and at similar levels as shown by western blot (Figure 6B). Moreover, all three lines exhibited similar global levels of H3K27me3 and Oct4 protein (Figure 6B). Interestingly, Jarid2 WT rescued ESC presented slight morphological differences, displaying more typical ESC colonies, when compared to Jarid2 KO or K116A mutant cells that tend to show a more flattened morphology (Figure 6C, left). They also formed fewer colonies when plated at low density as compared to the K116A rescued line (Figure 6C, right). Nonetheless, all the three ESC lines showed similar levels of alkaline phosphatase activity, comparable proliferation rates, and no major differences in gene expression (Figure 6D and day 0 in Figure 6F), thus confirming that Jarid2 is not necessary for ESC pluripotency and self-renewal (Landeira et al., 2010; Shen et al., 2009).


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 Is Required to Set Up Differentiation Programs in ESC(A) IF for Jarid2 and Oct4 in Jarid2 KO and Jarid2 WT or K116A rescued ESC.(B) WB with the indicated antibodies on nuclear extracts of the three ESC lines.(C) Left: colony morphology quantification of the indicated ESC. Values represent the percentage of alkaline-phosphatase-positive colonies showing a compact, flat, or spread morphology (mean ± SD, n ≥ 2). Right: number of methylene-blue-stained colonies (mean ± SD, n ≥ 2) detected 8 days after plating 100 cells per well.(D) Top: cell growth curve of the ESC over 4 days (mean ± SD, n = 3). Bottom: representative view of phosphatase alkaline staining.(E) WB probing nuclear extracts during ESC differentiation.(F) RNA transcript quantification through multiplexed-digital hybridization-based analysis (mean, n = 2). Heatmap representing the Log2-transformed median centered values. Top: messenger RNA classified by function. Bottom: transcripts from repeated sequences. Values are normalized around a set of housekeeping genes (ActB, Hprt1, Gapdh, Ppia, RplPO, and Rrm2). See also Figure S6.
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fig6: Jarid2 Methylation Is Required to Set Up Differentiation Programs in ESC(A) IF for Jarid2 and Oct4 in Jarid2 KO and Jarid2 WT or K116A rescued ESC.(B) WB with the indicated antibodies on nuclear extracts of the three ESC lines.(C) Left: colony morphology quantification of the indicated ESC. Values represent the percentage of alkaline-phosphatase-positive colonies showing a compact, flat, or spread morphology (mean ± SD, n ≥ 2). Right: number of methylene-blue-stained colonies (mean ± SD, n ≥ 2) detected 8 days after plating 100 cells per well.(D) Top: cell growth curve of the ESC over 4 days (mean ± SD, n = 3). Bottom: representative view of phosphatase alkaline staining.(E) WB probing nuclear extracts during ESC differentiation.(F) RNA transcript quantification through multiplexed-digital hybridization-based analysis (mean, n = 2). Heatmap representing the Log2-transformed median centered values. Top: messenger RNA classified by function. Bottom: transcripts from repeated sequences. Values are normalized around a set of housekeeping genes (ActB, Hprt1, Gapdh, Ppia, RplPO, and Rrm2). See also Figure S6.
Mentions: The results presented so far demonstrate that Jarid2 methylation is important for PRC2 activity in vitro and when artificially tethered to chromatin in a cellular context. To confirm the biological relevance of this regulation, we evaluated the function of Jarid2 methylation in ESC. We used ESC KO for Jarid2 (Shen et al., 2009) and generated cell lines rescued with either WT or K116A mutant forms of Jarid2. Since Jarid2 is dynamically regulated during cell differentiation, the rescues were performed using bacterial artificial chromosomes (BACs) in order to recapitulate the endogenous expression of the Jarid2 gene. Rescued ESC lines expressed Jarid2 homogeneously, with appropriate nuclear distributions as illustrated by IF (Figure 6A) and at similar levels as shown by western blot (Figure 6B). Moreover, all three lines exhibited similar global levels of H3K27me3 and Oct4 protein (Figure 6B). Interestingly, Jarid2 WT rescued ESC presented slight morphological differences, displaying more typical ESC colonies, when compared to Jarid2 KO or K116A mutant cells that tend to show a more flattened morphology (Figure 6C, left). They also formed fewer colonies when plated at low density as compared to the K116A rescued line (Figure 6C, right). Nonetheless, all the three ESC lines showed similar levels of alkaline phosphatase activity, comparable proliferation rates, and no major differences in gene expression (Figure 6D and day 0 in Figure 6F), thus confirming that Jarid2 is not necessary for ESC pluripotency and self-renewal (Landeira et al., 2010; Shen et al., 2009).

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