POLE3-POLE4 Is a Histone H3-H4 Chaperone that Maintains Chromatin Integrity during DNA Replication

摘要

class="head no_bottom_margin" id="sec1title">IntroductionDuring S phase of the cell cycle, accurate and processive replication of genomic DNA has to be coupled to duplication of the epigenetic information encoded in histones and their post-translational modifications (). For this to happen, chromatin must be disrupted ahead of the replication fork and restored in a timely and regulated fashion on sister chromatids, a process known as replication-coupled nucleosome assembly (, , ). This process relies on two different but interlocked mechanisms involving the recycling of parental histones and the deposition of newly synthesized ones.Coordination of these processes is particularly important for both the transmission of heterochromatic domains, such as those next to centromeres and telomeres, and the maintenance of cellular differentiation and identity (, , ). Furthermore, alteration of chromatin assembly has been linked to human genetic diseases, such as Wolf-Hirschhorn syndrome and congenital dyserythropoietic anemia type I (, ), as well as to acquired diseases such as cancer (). Changes in chromatin composition and structure also impact on aging (, ).The mechanisms that coordinate parental histone recycling and deposition of newly synthesized histones and how these function during leading- and lagging-strand replication remain to be deciphered. Several histone chaperones have been implicated in handling histones and participate in their transfer during DNA replication (). The histone chaperone CAF-1 (chromatin assembly factor 1) deposits new histones in a DNA synthesis-dependent manner (, , href="#bib62" rid="bib62" class=" bibr popnode">Verreault et al., 1996). With respect to recycling of parental histones, a role has been recently ascribed to the MCM2 component of the eukaryotic replicative helicase MCM2-7, which directly binds histones H3-H4 via a N-terminal domain (href="#bib28" rid="bib28" class=" bibr popnode">Ishimi et al., 2001, href="#bib16" rid="bib16" class=" bibr popnode">Foltman et al., 2013), together with the essential histone chaperone Asf1 (href="#bib23" rid="bib23" class=" bibr popnode">Groth et al., 2007b). Structural studies have revealed that MCM2 acts by shielding H3-H4 surfaces normally bound by DNA or histones H2A/H2B (href="#bib26" rid="bib26" class=" bibr popnode">Huang et al., 2015, href="#bib51" rid="bib51" class=" bibr popnode">Richet et al., 2015). The histone chaperone FACT (facilitates chromatin transcription), which is able to chaperone both H2A/H2B and H3/H4 histones, has been found to travel with the replisome in S. cerevisiae (href="#bib19" rid="bib19" class=" bibr popnode">Gambus et al., 2006) and is proposed to participate in chromatin dismantling/deposition at the fork (href="#bib16" rid="bib16" class=" bibr popnode">Foltman et al., 2013, href="#bib64" rid="bib64" class=" bibr popnode">Yang et al., 2016, href="#bib35" rid="bib35" class=" bibr popnode">Kurat et al., 2017). Recent studies have also shown that the single-strand binding protein RPA binds histones H3-H4 and promotes nucleosome assembly at the replication fork (href="#bib38" rid="bib38" class=" bibr popnode">Liu et al., 2017).Observations in budding and fission yeast have suggested a role for the leading-strand polymerase Polε in maintaining heterochromatin regions (href="#bib27" rid="bib27" class=" bibr popnode">Iida and Araki, 2004, href="#bib36" rid="bib36" class=" bibr popnode">Li et al., 2011); this function seems to be mainly dependent upon the smallest subunits of Polε, Dpb3 and Dpb4 (href="#bib27" rid="bib27" class=" bibr popnode">Iida and Araki, 2004, href="#bib25" rid="bib25" class=" bibr popnode">He et al., 2017). ΔDpb3 and ΔDpb4 yeast strains exhibit defective heterochromatin maintenance, which is also shared with strains lacking essential replisome-associated chaperones such as CAF-1, Asf1, and MCM2 HBD (histone binding domain) (href="#bib55" rid="bib55" class=" bibr popnode">Singer et al., 1998, href="#bib30" rid="bib30" class=" bibr popnode">Kaufman et al., 1997, href="#bib16" rid="bib16" class=" bibr popnode">Foltman et al., 2013). How Dpb3 and Dpb4 contribute to the maintenance of heterochromatin remains unclear.Here we show that the POLE3-POLE4 accessory subunits of mammalian Polε selectively bind to histones H3-H4 during replication-coupled nucleosome assembly. We define the mechanistic basis of POLE3-POLE4 binding and uncover an intrinsic chaperone activity toward H3-H4. In mammalian cells, depletion of POLE3 or POLE4 or removal of the C terminus of POLE3, which confers binding to H3-H4, directly impacts on nucleosome dynamics at the replication fork. Collectively, our work reveals mammalian POLE3-POLE4 as a replisome-associated histone H3-H4 chaperone that plays an important role in chromatin maintenance during DNA replication.