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Gelman and S

Gelman and S. developmental genes to keep up cell identity. They also repress repeated sequences such as major satellites and constitute an alternative state of BLZ945 pericentromeric constitutive heterochromatin at paternal chromosomes (pat\PCH) in mouse pre\implantation embryos. Amazingly, pat\PCH contains the histone H3.3 variant, which is absent from canonical PCH at maternal chromosomes, which is marked by histone H3 lysine 9 trimethylation (H3K9me3), HP1, and ATRX BLZ945 proteins. Here, we display that SUMO2\revised CBX2\comprising Polycomb Repressive Complex 1 (PRC1) recruits the H3.3\specific chaperone DAXX to pat\PCH, enabling H3.3 incorporation at these loci. Deficiency of or PRC1 parts and abrogates H3.3 incorporation, induces chromatin decompaction and breakage at PCH of exclusively paternal chromosomes, and causes their mis\segregation. Complementation assays display that DAXX\mediated H3.3 deposition is required for chromosome stability in early embryos. DAXX also regulates repression of PRC1 target genes during oogenesis and early embryogenesis. The study identifies a novel critical part for Polycomb in ensuring heterochromatin integrity and chromosome stability in mouse early development. and deficiency impaired the heterochromatin state at and function of centromeres (Morozov induces improved recruitment of cPCR1 to PcG target genes and their repression (Kang and results in loss of binding of DAXX and H3.3 occupancy at pat\PCH. The two SUMO\interacting motifs (SIMs) of DAXX are required for its association with pat\PCH implying a role for SUMOylation in DAXX chromatin focusing on to these loci. Accordingly, mutation of specific residues in CBX2, which impair its SH3RF1 SUMOylation, prevent DAXX focusing on to PCH. Finally, we demonstrate that loss of H3.3 at pat\PCH upon knockout induces chromatin decompaction and breakage at PCH of exclusively paternal chromosomes and causes their mis\segregation. We display that H3.3 deposition by DAXX is required for chromosome stability in early embryos. Therefore, we determine a novel pathway and part for SUMOylation and Polycomb in ensuring chromatin integrity. Genome\wide transcriptional analysis demonstrates regulates repression of PRC1 target genes in oocytes and 2\cell embryos. Our data suggest a regulatory function of the novel CBX2/cPRC1??SUMO2??DAXX??H3.3 pathway in PRC1\mediated gene silencing during mouse development. Results The histone variant H3.3 is incorporated into pat\PCH prior to the first round of DNA replication The paternal genome undergoes extensive chromatin remodeling shortly after fertilization, with the alternative of sperm\born protamines by maternally provided histones. The remodeling process happens many hours before the 1st round of replication arguing for nucleosome deposition onto the paternal DNA template. To monitor the timing of incorporation of histone proteins at pat\PCH in mouse zygotes, we microinjected mRNAs encoding for EGFP\tagged H3.2 and mCherry\tagged H3.3 proteins into metaphase II (M\II) oocytes prior to their activation by intracytoplasmic sperm injection (ICSI). We monitored the localization of the tagged histones by fluorescence spinning\disk live microscopy in fertilized embryos (Fig?EV1A; [Link], [Link], [Link]). As reported previously (Akiyama is required for H3.3 deposition in the decondensing sperm (Lin conditionally deficient or siRNA\treated mouse zygotes. mRNA transcripts and siRNAs were microinjected in MII\arrested oocytes, which were consequently fertilized by injection of sperm (ICSI). Still images of time\lapse imaging of 1st cell cycle showing temporal and spatial dynamics of H3. 3\mCherry and H3.3A87S/I89V/G90M\EGFP proteins in crazy\type zygotes ((and but also additional H3.3 chaperones like and are abundantly indicated (Fig?EV1D, and Park (Arakawa (HMT lacking both H3K9me3 and HP1 at PCH (Fig?EV1E and F) (Peters by siRNA injection (Fig.?1D) and investigated ATRX localization in late\stage zygotes. While the ATRX transmission at euchromatin and mat\PCH was unaffected, ATRX was specifically lost from pat\PCH (Fig?1D), demonstrating that ATRX association with pat\PCH requires DAXX. In contrast, maternal deficiency of the ATRX protein, resulting from conditional deletion of the gene during oogenesis, caused complete loss of DAXX protein from mat\PCH only, while DAXX levels at BLZ945 pat\PCH were unaffected (Fig?1E). Therefore, these genetic experiments demonstrate unambiguously that DAXX localization at pat\PCH is definitely upstream of ATRX, while it is definitely downstream at mat\PCH. Canonical PRC1 associates genetically and biochemically with DAXX We next investigated whether PRC2 or cPRC1 settings DAXX localization to pat\PCH. A earlier study implicated lysine 27 of H3.3 (H3.3K27) in formation and function of PCH in early embryos (Santenard and/or the paralog did not impact DAXX localization at pat\PCH (Fig?2A) excluding PRC2 and H3K27me3 while key determinants of DAXX recruitment and function in mouse zygotes. Open in a separate window Number 2 Focusing on of DAXX to pat\PCH is dependent on PRC1 IF of DAXX and.