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. 2016 Nov 28:7:13661.
doi: 10.1038/ncomms13661.

Jarid2 binds mono-ubiquitylated H2A lysine 119 to mediate crosstalk between Polycomb complexes PRC1 and PRC2

Sarah Cooper  1 , Anne Grijzenhout  1 , Elizabeth Underwood  1 , Katia Ancelin  2 , Tianyi Zhang  1 , Tatyana B Nesterova  1 , Burcu Anil-Kirmizitas  1 , Andrew Bassett  3 , Susanne M Kooistra  4   5 , Karl Agger  4   5 , Kristian Helin  4   5   6 , Edith Heard  2 , Neil Brockdorff  1
Affiliations

Jarid2 binds mono-ubiquitylated H2A lysine 119 to mediate crosstalk between Polycomb complexes PRC1 and PRC2

Sarah Cooper et al. Nat Commun. .

Abstract

The Polycomb repressive complexes PRC1 and PRC2 play a central role in developmental gene regulation in multicellular organisms. PRC1 and PRC2 modify chromatin by catalysing histone H2A lysine 119 ubiquitylation (H2AK119u1), and H3 lysine 27 methylation (H3K27me3), respectively. Reciprocal crosstalk between these modifications is critical for the formation of stable Polycomb domains at target gene loci. While the molecular mechanism for recognition of H3K27me3 by PRC1 is well defined, the interaction of PRC2 with H2AK119u1 is poorly understood. Here we demonstrate a critical role for the PRC2 cofactor Jarid2 in mediating the interaction of PRC2 with H2AK119u1. We identify a ubiquitin interaction motif at the amino-terminus of Jarid2, and demonstrate that this domain facilitates PRC2 localization to H2AK119u1 both in vivo and in vitro. Our findings ascribe a critical function to Jarid2 and define a key mechanism that links PRC1 and PRC2 in the establishment of Polycomb domains.

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Figures

Figure 1
Figure 1. JARID2 is required for recruitment of PRC2 to H2AK119u1 modified chromatin.
(a) Schematic of the experiment. The MBD localizes to high meCpG regions including PCH. The minimal E3 ligase fusion protein (RPCD) catalyses H2AK119u1 but does not interact with PRC1 or PRC2. (b) Immunofluorescence staining of WT and Aebp2tr/tr mESCs. Scale bar, 5 μm. Arrowhead indicates a PCH focus. (c) Quantification of H3K27me3-positive PCH domains shown in b. (d,e) As (b,c) but for Jarid2 WT and Jarid2 KO mESCs. A minimum of 300 cells were counted in three biological repeats. Error bars indicate s.d.
Figure 2
Figure 2. JARID2 is required for establishment of H3K27me3 at PCH domains upon loss of DNA methylation.
(a) Schematic of the experimental set-up. DNA methylation (filled lollipops) was removed by tamoxifen induction of conditional Dnmt1F/F mESCs and culturing for 6–12 days. In response to the loss of DNA methylation PRC1 (H2AK119u1) and PRC2 (H3K27me3) complexes localize to PCH (major satellites). (b) Immunofluorescence staining with H2AK119u1 (left) and quantification of PCH domains (right) of Dnmt1−/− and Dnmt−/− Jarid2 KO mESCs. (c) As (b), except staining and quantification for H3K27me3. A minimum of 300 cells were counted in three biological repeats. Error bars indicate s.d. Scale bars, 5 μm.
Figure 3
Figure 3. A ubiquitin binding motif in the Jarid2 N-terminus mediates interaction with H2AK119u1.
(a) Schematic showing full length JARID2 and complementation constructs, UIM, ubiquitin interaction motif; TRD, transcription repressive domain; JmjN, JumonjiN; JmjC, JumonjiC; and ZF, zinc finger domain. (b) Levels of restoration of H3K27me3 in response to H2AK119u1 after complementation by the constructs in a. (c) Consensus UIM sequence and alignment of single-sided UIMs with sequence from Jarid2 N-terminus. (d) JARID2 UIM peptide interacts with ubiquitin. Coumarin-JARID2 24–43 binds to WT ubiquitin (yellow) more strongly that I44A ubiquitin (red) as monitored by fluorescence polarization (n=3). (e) Co-localization of EZH2 with Xist-Bgl-mCherry domains in wild type, Jarid2 null mESCs and in mESC cell lines complemented with the indicated constructs. A minimum of 200 Xist-Bgl-mCherry-positive cells were counted in three biological repeats in all experiments. Error bars indicate s.d.
Figure 4
Figure 4. JARID2 N-terminus mediates direct interaction with H2AK119u1-modified nucleosomes
(a) Immunoblot of nucleosomal pull downs from nuclear extract. Total nuclear extract (input) and pull down using streptavidin beads, unmodified nucleosomes or nucleosomes modified by H2AK119u1/H2AK119u1(I44A) were probed with the indicated antibodies using extracts from WT cells (left) or JARID2 KO mESCs (right). Full blots are shown in Supplementary Fig. 9. (b) JARID2 1–530 binds to H2AK119u1-modified mononucleosomes (yellow) more tightly than unmodified (blue) or H2AK199u1(I44A) (red) as monitored by biolayer interferometry (n=3).
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