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The centromeric nucleosome Centromeres are epigenetically marked by the assembly of nucleosomes containing CENP-A, a centromere-specific histone H3 variant. Tachiwana et al . report the crystal structure of the human centromeric nucleosome bound to DNA. They find a canonical arrangement of an octamer of histone proteins with DNA wrapped in a left-handed superhelix. There is flexibility in the DNA regions at the entrance and exit of the nucleosome, and a loop in CENP-A may help to stabilize its incorporation into centromeric chromatin. As the first view of the CENP-A-containing nucleosome, the structure helps to clarify various models that have been debated in the literature. In eukaryotes, accurate chromosome segregation during mitosis and meiosis is coordinated by kinetochores, which are unique chromosomal sites for microtubule attachment 1 , 2 . Centromeres specify the kinetochore formation sites on individual chromosomes, and are epigenetically marked by the assembly of nucleosomes containing the centromere-specific histone H3 variant, CENP-A 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 . Although the underlying mechanism is unclear, centromere inheritance is probably dictated by the architecture of the centromeric nucleosome. Here we report the crystal structure of the human centromeric nucleosome containing CENP-A and its cognate α-satellite DNA derivative (147 base pairs). In the human CENP-A nucleosome, the DNA is wrapped around the histone octamer, consisting of two each of histones H2A, H2B, H4 and CENP-A, in a left-handed orientation. However, unlike the canonical H3 nucleosome, only the central 121 base pairs of the DNA are visible. The thirteen base pairs from both ends of the DNA are invisible in the crystal structure, and the αN helix of CENP-A is shorter than that of H3, which is known to be important for the orientation of the DNA ends in the canonical H3 nucleosome 13 . A structural comparison of the CENP-A and H3 nucleosomes revealed that CENP-A contains two extra amino acid residues (Arg 80 and Gly 81) in the loop 1 region, which is completely exposed to the solvent. Mutations of the CENP-A loop 1 residues reduced CENP-A retention at the centromeres in human cells. Therefore, the CENP-A loop 1 may function in stabilizing the centromeric chromatin containing CENP-A, possibly by providing a binding site for trans -acting factors. The structure provides the first atomic-resolution picture of the centromere-specific nucleosome.

Heterochromatin affects genome function at many levels. It enables heritable gene repression, maintains chromosome integrity and provides mechanical rigidity to the nucleus 1 , 2 . These diverse functions are proposed to arise in part from compaction of the underlying chromatin 2 . A major type of heterochromatin contains at its core the complex formed between HP1 proteins and chromatin that is methylated on histone H3, lysine 9 (H3K9me). HP1 is proposed to use oligomerization to compact chromatin into phase-separated condensates 3 – 6 . Yet, how HP1-mediated phase separation relates to chromatin compaction remains unclear. Here we show that chromatin compaction by the Schizosaccharomyces pombe HP1 protein Swi6 results in phase-separated liquid condensates. Unexpectedly, we find that Swi6 substantially increases the accessibility and dynamics of buried histone residues within a nucleosome. Restraining these dynamics impairs compaction of chromatin into liquid droplets by Swi6. Our results indicate that Swi6 couples its oligomerization to the phase separation of chromatin by a counterintuitive mechanism, namely the dynamic exposure of buried nucleosomal regions. We propose that such reshaping of the octamer core by Swi6 increases opportunities for multivalent interactions between nucleosomes, thereby promoting phase separation. This mechanism may more generally drive chromatin organization beyond heterochromatin. The S. pombe HP1 protein Swi6 couples chromatin compaction to phase separation by dynamically exposing buried histone residues within nucleosomes.

In the eukaryotic nucleus, DNA is packaged in the form of nucleosomes, each of which comprises about 147 base pairs of DNA wrapped around a histone protein octamer. The position and histone composition of nucleosomes is governed by ATP-dependent chromatin remodellers 1 – 3 such as the 15-subunit INO80 complex 4 . INO80 regulates gene expression, DNA repair and replication by sliding nucleosomes, the exchange of histone H2A.Z with H2A, and the positioning of + 1 and −1 nucleosomes at promoter DNA 5 – 8 . The structures and mechanisms of these remodelling reactions are currently unknown. Here we report the cryo-electron microscopy structure of the evolutionarily conserved core of the INO80 complex from the fungus Chaetomium thermophilum bound to a nucleosome, at a global resolution of 4.3 Å and with major parts at 3.7 Å. The INO80 core cradles one entire gyre of the nucleosome through multivalent DNA and histone contacts. An Rvb1/Rvb2 AAA + ATPase heterohexamer is an assembly scaffold for the complex and acts as a ‘stator’ for the motor and nucleosome-gripping subunits. The Swi2/Snf2 ATPase motor binds to nucleosomal DNA at superhelical location −6, unwraps approximately 15 base pairs, disrupts the H2A–DNA contacts and is poised to pump entry DNA into the nucleosome. Arp5 and Ies6 bind superhelical locations −2 and −3 to act as a counter grip for the motor, on the other side of the H2A–H2B dimer. The Arp5 insertion domain forms a grappler element that binds the nucleosome dyad, connects the Arp5 actin-fold and entry DNA over a distance of about 90 Å and packs against histone H2A–H2B near the ‘acidic patch’. Our structure together with biochemical data 8 suggests a unified mechanism for nucleosome sliding and histone editing by INO80. The motor is part of a macromolecular ratchet, persistently pumping entry DNA across the H2A–H2B dimer against the Arp5 grip until a large nucleosome translocation step occurs. The transient exposure of H2A–H2B by motor activity as well as differential recognition of H2A.Z and H2A may regulate histone exchange. Cryo-electron microscopy structures of the evolutionarily conserved core of a fungal INO80 complex bound to the nucleosomal substrate reveal the mechanism underlying nucleosome sliding and histone editing used by this ATP-dependent chromatin remodeller.

Histone demethylase activity of PHF8 Mutations in the PHF8 gene, which encodes the plant homeo domain (PHD) finger protein 8, are connected to X-linked mental retardation associated with cleft lip and cleft palate. Two groups now report that the PHF8 protein is a histone demethylase with activity against H4K20me1 (histone H4 lysine 20). Qi et al . report a role for PHF8 in regulating gene expression, as well as in neuronal cell survival and craniofacial development in zebrafish. The results suggest there may be a link between histone methylation dynamics and X-linked mental retardation. Liu et al . show that PHF8 is linked to two distinct events during cell-cycle progression. PHF8 is recruited to the promoters of G1/S-phase genes where it removes H4K20me1 and contributes to gene activation, whereas dissociation of PHF8 from chromatin in prophase allows H4K20me1 to accumulate during mitosis. These authors show that the JmjC domain-containing protein PHF8 has histone demethylase activity against H4K20me1 and is linked to two distinct events during cell cycle progression. PHF8 is recruited to the promoters of genes involved in the G1–S phase transition, where it removes H4K20me1 and contributes to gene activation, whereas dissociation of PHF8 from chromatin in prophase allows H4K20me1 to accumulate during mitosis. While reversible histone modifications are linked to an ever-expanding range of biological functions 1 , 2 , 3 , 4 , 5 , the demethylases for histone H4 lysine 20 and their potential regulatory roles remain unknown. Here we report that the PHD and Jumonji C (JmjC) domain-containing protein, PHF8, while using multiple substrates, including H3K9me1/2 and H3K27me2, also functions as an H4K20me1 demethylase. PHF8 is recruited to promoters by its PHD domain based on interaction with H3K4me2/3 and controls G1–S transition in conjunction with E2F1, HCF-1 (also known as HCFC1) and SET1A (also known as SETD1A), at least in part, by removing the repressive H4K20me1 mark from a subset of E2F1-regulated gene promoters. Phosphorylation-dependent PHF8 dismissal from chromatin in prophase is apparently required for the accumulation of H4K20me1 during early mitosis, which might represent a component of the condensin II loading process. Accordingly, the HEAT repeat clusters in two non-structural maintenance of chromosomes (SMC) condensin II subunits, N-CAPD3 and N-CAPG2 (also known as NCAPD3 and NCAPG2, respectively), are capable of recognizing H4K20me1, and ChIP-Seq analysis demonstrates a significant overlap of condensin II and H4K20me1 sites in mitotic HeLa cells. Thus, the identification and characterization of an H4K20me1 demethylase, PHF8, has revealed an intimate link between this enzyme and two distinct events in cell cycle progression.

53BP1 (also called TP53BP1) is a chromatin-associated factor that promotes immunoglobulin class switching and DNA double-strand-break (DSB) repair by non-homologous end joining. To accomplish its function in DNA repair, 53BP1 accumulates at DSB sites downstream of the RNF168 ubiquitin ligase. How ubiquitin recruits 53BP1 to break sites remains unknown as its relocalization involves recognition of histone H4 Lys 20 (H4K20) methylation by its Tudor domain. Here we elucidate how vertebrate 53BP1 is recruited to the chromatin that flanks DSB sites. We show that 53BP1 recognizes mononucleosomes containing dimethylated H4K20 (H4K20me2) and H2A ubiquitinated on Lys 15 (H2AK15ub), the latter being a product of RNF168 action on chromatin. 53BP1 binds to nucleosomes minimally as a dimer using its previously characterized methyl-lysine-binding Tudor domain and a carboxy-terminal extension, termed the ubiquitination-dependent recruitment (UDR) motif, which interacts with the epitope formed by H2AK15ub and its surrounding residues on the H2A tail. 53BP1 is therefore a bivalent histone modification reader that recognizes a histone ‘code’ produced by DSB signalling. This study shows that 53BP1 recruitment to sites of DNA damage involves dual recognition of H4K20me2 and H2AK15 histone ubiquitination; the ubiquitin mark and the surrounding epitope on H2A are read by a region of 53BP1 designated the ubiquitination-dependent recruitment motif. Recruiting 53BP1 protein to DNA damage sites The key DNA damage response protein 53BP1 acts by binding to chromatin at the site of a double-strand break. Previous studies suggested that 53BP1 acts after a ubiquitination event promoted by RNF168, although its recruitment to breaks was thought to depend only on histone H4K20 methylation. Daniel Durocher and colleagues now show that 53BP1 recruitment involves the recognition of both H4K20me2 and histone H2AK15 ubiquitination. The ubiquitin mark, and the surrounding context on histone H2A, are read by a region of 53BP1 that the authors designate the ubiquitination-dependent recruitment motif.

In eukaryotes, DNA is packed onto nucleosomes. For transcription factors and other proteins to gain access to DNA, nucleosomes must be moved out of the way, or “remodeled”—but not disassembled. Nucleosomes are composed of histone protein octamers, the cores of which have generally been considered to be fairly rigid. Sinha et al. used nuclear magnetic resonance and protein cross-linking to show that one of the enzyme complexes that remodel nucleosomes, SNF2h, is able to distort the histone octamer (see the Perspective by Flaus and Owen-Hughes). Nucleosome deformation was important for this remodeler to be able to slide nucleosomes out of the way. Science , this issue p. 10.1126/science.aaa3761 ; see also p. 245 The nucleosome histone octamer can be deformed by a nucleosome remodeling enzyme to slide nucleosomes out of the way. Adenosine 5′-triphosphate (ATP)–dependent chromatin remodeling enzymes play essential biological roles by mobilizing nucleosomal DNA. Yet, how DNA is mobilized despite the steric constraints placed by the histone octamer remains unknown. Using methyl transverse relaxation–optimized nuclear magnetic resonance spectroscopy on a 450-kilodalton complex, we show that the chromatin remodeler, SNF2h, distorts the histone octamer. Binding of SNF2h in an activated ATP state changes the dynamics of buried histone residues. Preventing octamer distortion by site-specific disulfide linkages inhibits nucleosome sliding by SNF2h while promoting octamer eviction by the SWI-SNF complex, RSC. Our findings indicate that the histone core of a nucleosome is more plastic than previously imagined and that octamer deformation plays different roles based on the type of chromatin remodeler. Octamer plasticity may contribute to chromatin regulation beyond ATP-dependent remodeling.

Methylation of DNA and lysine 9 of histone H3 (H3K9) are well‐conserved epigenetic marks for transcriptional silencing. Although H3K9 methylation directs DNA methylation in filamentous fungi and plants, this pathway has not been corroborated in mammals. G9a and GLP/Eu‐HMTase1 are two‐related mammalian lysine methyltransferases and a G9a/GLP heteromeric complex regulates H3K9 methylation of euchromatin. To elucidate the function of G9a/GLP‐mediated H3K9 methylation in the regulation of DNA methylation and transcriptional silencing, we characterized ES cells expressing catalytically inactive mutants of G9a and/or GLP. Interestingly, in ES cells expressing a G9a‐mutant/GLP complex that does not rescue global H3K9 methylation, G9a/GLP‐target genes remain silent. The CpG sites of the promoter regions of these genes were hypermethylated in such mutant ES cells, but hypomethylated in G9a‐ or GLP ‐KO ES cells. Treatment with a DNA methyltransferase inhibitor reactivates these G9a/GLP‐target genes in ES cells expressing catalytically inactive G9a/GLP proteins, but not the wild‐type proteins. This is the first clear evidence that G9a/GLP suppresses transcription by independently inducing both H3K9 and DNA methylation.

The interplay of histone acetylation and RNA polymerase II activity is investigated using fluorescence microscopy; acetylation of H3 at Lys 27 enhances the recruitment of a transcriptional activator and accelerates the transition of RNA polymerase II from initiation to elongation, thus indicating that histone acetylation has a causal effect on two distinct steps in transcription activation. Histone modification in a living cell Post-translational modifications to histone proteins have an important role in gene regulation, but it remains unclear if these marks are active regulators of transcription or downstream consequences. Here, Tim Stasevich et al . investigate the dynamic interplay of histone acetylation and RNA polymerase II activity at high temporal resolution in single living cells using fluorescence microscopy. Acetylation of histone H3 at Lys27 at an active gene locus enhances the recruitment of a transcriptional activator and accelerates the transition of RNA polymerase II from initiation to elongation. These findings indicate that histone acetylation has a causal effect on two distinct steps in transcription activation. In eukaryotic cells, post-translational histone modifications have an important role in gene regulation. Starting with early work on histone acetylation 1 , a variety of residue-specific modifications have now been linked to RNA polymerase II (RNAP2) activity 2 , 3 , but it remains unclear if these markers are active regulators of transcription or just passive byproducts 4 , 5 . This is because studies have traditionally relied on fixed cell populations, meaning temporal resolution is limited to minutes at best, and correlated factors may not actually be present in the same cell at the same time. Complementary approaches are therefore needed to probe the dynamic interplay of histone modifications and RNAP2 with higher temporal resolution in single living cells 2 , 5 , 6 . Here we address this problem by developing a system to track residue-specific histone modifications and RNAP2 phosphorylation in living cells by fluorescence microscopy. This increases temporal resolution to the tens-of-seconds range. Our single-cell analysis reveals histone H3 lysine-27 acetylation at a gene locus can alter downstream transcription kinetics by as much as 50%, affecting two temporally separate events. First acetylation enhances the search kinetics of transcriptional activators, and later the acetylation accelerates the transition of RNAP2 from initiation to elongation. Signatures of the latter can be found genome-wide using chromatin immunoprecipitation followed by sequencing. We argue that this regulation leads to a robust and potentially tunable transcriptional response.

Nucleosomes containing the histone H3 variant CENP-A are the epigenetic mark of centromeres, the kinetochore assembly sites required for chromosome segregation. HJURP is the CENP-A chaperone, which associates with Mis18α, Mis18β, and M18BP1 to target centromeres and deposit new CENP-A. How these proteins interact to promote CENP-A deposition remains poorly understood. Here we show that two repeats in human HJURP proposed to be functionally distinct are in fact interchangeable and bind concomitantly to the 4:2:2 Mis18α:Mis18β:M18BP1 complex without dissociating it. HJURP binds CENP-A:H4 dimers, and therefore assembly of CENP-A:H4 tetramers must be performed by two Mis18αβ:M18BP1:HJURP complexes, or by the same complex in consecutive rounds. The Mis18α N-terminal tails blockade two identical HJURP-repeat binding sites near the Mis18αβ C-terminal helices. These were identified by photo-cross-linking experiments and mutated to separate Mis18 from HJURP centromere recruitment. Our results identify molecular underpinnings of eukaryotic chromosome inheritance and shed light on how centromeres license CENP-A deposition. The CENP-A chaperone HJURP associates with Mis18α, Mis18β, and M18BP1 to target centromeres and deposit new CENP-A. Here the authors provide evidence that two repeats in human HJURP previously proposed to be functionally distinct are interchangeable and bind concomitantly to the 4:2:2 Mis18α:Mis18β:M18BP1 complex without dissociating it.

Osterix (Osx) is an osteoblast‐specific transcription factor required for osteoblast differentiation and bone formation. Osx null mice develop a normal cartilage skeleton but fail to form bone and to express osteoblast‐specific marker genes. To better understand the control of transcriptional regulation by Osx, we identified Osx‐interacting proteins using proteomics approaches. Here, we report that a Jumonji C (JmjC)‐domain containing protein, called NO66, directly interacts with Osx and inhibits Osx‐mediated promoter activation. The knockdown of NO66 in preosteoblast cells triggered accelerated osteoblast differentiation and mineralization, and markedly stimulated the expression of Osx target genes. A JmjC‐dependent histone demethylase activity was exhibited by NO66, which was specific for both H3K4me and H3K36me in vitro and in vivo , and this activity was needed for the regulation of osteoblast‐specific promoters. During BMP‐2‐induced differentiation of preosteoblasts, decreased NO66 occupancy correlates with increased Osx occupancy at Osx‐target promoters. Our results indicate that interactions between NO66 and Osx regulate Osx‐target genes in osteoblasts by modulating histone methylation states.