Nucleosomal Histone H3 Research Articles

Cryo-EM structure of the human Sirtuin 6-nucleosome complex.

Sirtuin 6 (SIRT6) is a multifaceted protein deacetylase/deacylase and a major target for small-molecule modulators of longevity and cancer. In the context of chromatin, SIRT6 removes acetyl groups from histone H3 in nucleosomes, but the molecular basis for its nucleosomal substrate preference is unknown. Our cryo-electron microscopy structure of human SIRT6 in complex with the nucleosome shows that the catalytic domain of SIRT6 pries DNA from the nucleosomal entry-exit site and exposes the histone H3 N-terminal helix, while the SIRT6 zinc-binding domain binds to the histone acidic patch using an arginine anchor. In addition, SIRT6 forms an inhibitory interaction with the C-terminal tail of histone H2A. The structure provides insights into how SIRT6 can deacetylate both H3 K9 and H3 K56.

The Arabidopsis NuA4 histone acetyltransferase complex is required for chlorophyll biosynthesis and photosynthesis.

Although two Enhancer of Polycomb-like proteins, EPL1A and EPL1B (EPL1A/B), are known to be conserved and characteristic subunits of the NuA4-type histone acetyltransferase complex in Arabidopsis thaliana, the biological function of EPL1A/B and the mechanism by which EPL1A/B function in the complex remain unknown. Here, we report that EPL1A/B are required for the histone acetyltransferase activity of the NuA4 complex on the nucleosomal histone H4 in vitro and for the enrichment of histone H4K5 acetylation at thousands of protein-coding genes in vivo. Our results suggest that EPL1A/B are required for linking the NuA4 catalytic subunits HISTONE ACETYLTRANSFERASE OF THE MYST FAMILY 1（HAM1) and HAM2 with accessory subunits in the NuA4 complex. EPL1A/B function redundantly in regulating plant development especially in chlorophyll biosynthesis and de-etiolation. The EPL1A/B-dependent transcription and H4K5Ac are enriched at genes involved in chlorophyll biosynthesis and photosynthesis. We also find that EAF6, another characteristic subunit of the NuA4 complex, contributes to de-etiolation. These results suggest that the Arabidopsis NuA4 complex components function as a whole to mediate histone acetylation and transcriptional activation specifically at light-responsive genes and are critical for photomorphogenesis.

Structural basis of the regulation of the normal and oncogenic methylation of nucleosomal histone H3 Lys36 by NSD2

Dimethylated histone H3 Lys36 (H3K36me2) regulates gene expression, and aberrant H3K36me2 upregulation, resulting from either the overexpression or point mutation of the dimethyltransferase NSD2, is found in various cancers. Here we report the cryo-electron microscopy structure of NSD2 bound to the nucleosome. Nucleosomal DNA is partially unwrapped, facilitating NSD2 access to H3K36. NSD2 interacts with DNA and H2A along with H3. The NSD2 autoinhibitory loop changes its conformation upon nucleosome binding to accommodate H3 in its substrate-binding cleft. Kinetic analysis revealed that two oncogenic mutations, E1099K and T1150A, increase NSD2 catalytic turnover. Molecular dynamics simulations suggested that in both mutants, the autoinhibitory loop adopts an open state that can accommodate H3 more often than the wild-type. We propose that E1099K and T1150A destabilize the interactions that keep the autoinhibitory loop closed, thereby enhancing catalytic turnover. Our analyses guide the development of specific inhibitors of NSD2.

Prognostic Value and Immunological Role of MORF4-Related Gene-Binding Protein in Human Cancers.

MORF4-related gene-binding protein (MRGBP) is the subunit of the NuA4 histone acetyltransferase complex which is involved in transcriptional activation of select genes principally by acetylation of nucleosomal histones H4 and H2A. Much of the research indicated an oncogenic role of MRGBP in the development of cancers. However, it is still unknown the role MRGBP plays in human cancers, which deserves further exploration. In this research, the expression profile, prognostic value of MRGBP, and the relationship between MRGBP and immune infiltration were explored in 33 types of cancer. The differences in MRGBP expression in tumor and normal tissues were explored using data from The Cancer Genome Atlas, Gene Expression Omnibus and ONCOMINE. Analysis of the association between MRGBP and prognosis using Kaplan-Meier survival curve and COX analysis. The data of Tumor mutational burden (TMB), microsatellite instability (MSI) from TCGA. The relationship Between MRGBP expression and immunity was analyzed using the ESTIMATE algorithm and CIBERSORT. Furthermore, we explored MRGBP expression and the relationship between MRGBP expression and macrophage infiltration using immunohistochemical analysis in lower grade glioma (LGG). Our results revealed that MRGBP was highly expressed in most cancer tissues compared with normal tissues. Tumors with increased MRGBP expression had a high clinicopathologic stage and poor prognosis. The expression of MRGBP was closely related to the TMB, MSI. We also found a significant negative correlation between MRGBP expression and stromal scores and immune scores in various types of cancer. Furthermore, MRGBP expression was associated with a variety of immune cells including B cells, NK cells, T cells, and macrophages. LGG and LIHC was selected as representative cancer types for further study, the results of immunohistochemistry indicated that the protein levels of MRGBP were significantly elevated in tumor tissues. Moreover, our LIHC data analysis showed that patients with high MRGBP expression were associated with short survival rates and MRGBP was a risk factor to determine OS. Immunohistochemistry also confirmed that M0 macrophage infiltration in the MRGBP-high group significantly increased. In conclusion, these results reveal that MRGBP can serve as a potential prognostic biomarker and it plays an important role in tumor immune infiltration in various tumors, especially in LGG and LIHC.

Structural basis of nucleosomal histone H4 lysine 20 methylation by SET8 methyltransferase.

SET8 is solely responsible for histone H4 lysine-20 (H4K20) monomethylation, which preferentially occurs in nucleosomal H4. However, the underlying mechanism by which SET8 specifically promotes the H4K20 monomethylation in the nucleosome has not been elucidated. Here, we report the cryo-EM structures of the human SET8-nucleosome complexes with histone H3 and the centromeric H3 variant, CENP-A. Surprisingly, we found that the overall cryo-EM structures of the SET8-nucleosome complexes are substantially different from the previous crystal structure models. In the complexes with H3 and CENP-A nucleosomes, SET8 specifically binds the nucleosomal acidic patch via an arginine anchor, composed of the Arg188 and Arg192 residues. Mutational analyses revealed that the interaction between the SET8 arginine anchor and the nucleosomal acidic patch plays an essential role in the H4K20 monomethylation activity. These results provide the groundwork for understanding the mechanism by which SET8 specifically accomplishes the H4K20 monomethylation in the nucleosome.

Pilocarpine-induced seizures associate with modifications of LSD1/CoREST/HDAC1/2 epigenetic complex and repressive chromatin in mice hippocampus

Epilepsy is a neurological disorder of genetic or environmental origin characterized by recurrent spontaneous seizures. A rodent model of temporal lobe epilepsy is induced by a single administration of pilocarpine, a non-selective cholinergic muscarinic receptor agonist. The molecular changes associated with pilocarpine-induced seizures are still poorly described. Epigenetic multiprotein complexes that regulate gene expression by changing the structure of chromatin impose transcriptional memories. Among the epigenetic enzymes relevant to the epileptogenic process is lysine-specific demethylase 1 (LSD1, KDM1A), which regulates the expression of genes that control neuronal excitability. LSD1 forms complexes with the CoREST family of transcriptional corepressors, which are molecular bridges that bring HDAC1/2 and LSD1 enzymes to deacetylate and demethylate the tail of nucleosomal histone H3. To test the hypothesis that LSD1-complexes are involved in initial modifications associated with pilocarpine-induced epilepsy, we studied the expression of main components of LSD1-complexes and the associated epigenetic marks on isolated neurons and the hippocampus of pilocarpine-treated mice. Using a single injection of 300 mg/kg of pilocarpine and after 24 h, we found that protein levels of LSD1, CoREST2, and HDAC1/2 increased, while CoREST1 decreased in the hippocampus. In addition, we observed increased histone H3 lysine 9 di- and trimethylation (H3K9me2/3) and decreased histone H3 lysine 4 di and trimethylation (H3K4me2/3). Similar findings were observed in cultured hippocampal neurons and HT-22 hippocampal cell line treated with pilocarpine. In conclusion, our data show that muscarinic receptor activation by pilocarpine induces a global repressive state of chromatin and prevalence of LSD1-CoREST2 epigenetic complexes, modifications that could underlie the pathophysiological processes leading to epilepsy.

Dual histone methyl reader ZCWPW1 facilitates repair of meiotic double strand breaks in male mice.

Meiotic crossovers result from homology-directed repair of DNA double-strand breaks (DSBs). Unlike yeast and plants, where DSBs are generated near gene promoters, in many vertebrates DSBs are enriched at hotspots determined by the DNA binding activity of the rapidly evolving zinc finger array of PRDM9 (PR domain zinc finger protein 9). PRDM9 subsequently catalyzes tri-methylation of lysine 4 and lysine 36 of Histone H3 in nearby nucleosomes. Here, we identify the dual histone methylation reader ZCWPW1, which is tightly co-expressed during spermatogenesis with Prdm9, as an essential meiotic recombination factor required for efficient repair of PRDM9-dependent DSBs and for pairing of homologous chromosomes in male mice. In sum, our results indicate that the evolution of a dual histone methylation writer/reader (PRDM9/ZCWPW1) system in vertebrates remodeled genetic recombination hotspot selection from an ancestral static pattern near genes towards a flexible pattern controlled by the rapidly evolving DNA binding activity of PRDM9.

Dynamics of the nucleosomal histone H3 N-terminal tail revealed by high precision single-molecule FRET.

Chromatin compaction and gene accessibility are orchestrated by assembly and disassembly of nucleosomes. Although the disassembly process was widely studied, little is known about the structure and dynamics of the disordered histone tails, which play a pivotal role for nucleosome integrity. This is a gap filling experimental FRET study from the perspective of the histone H3 N-terminal tail (H3NtT) of reconstituted mononucleosomes. By systematic variation of the labeling positions we monitored the motions of the H3NtT relative to the dyad axis and linker DNA. Single-molecule FRET unveiled that H3NtTs do not diffuse freely but follow the DNA motions with multiple interaction modes with certain permitted dynamic transitions in the μs to ms time range. We also demonstrate that the H3NtT can allosterically sense charge-modifying mutations within the histone core (helix α3 of histone H2A (R81E/R88E)) resulting in increased dynamic transitions and lower rate constants. Those results complement our earlier model on the NaCl induced nucleosome disassembly as changes in H3NtT configurations coincide with two major steps: unwrapping of one linker DNA and weakening of the internal DNA - histone interactions on the other side. This emphasizes the contribution of the H3NtT to the fine-tuned equilibrium between overall nucleosome stability and DNA accessibility.

Multiple Interaction Modes of the Nucleosomal Histone H3 N-Terminal Tail Revealed by High Precision Single-Molecule FRET

Multiple Interaction Modes of the Nucleosomal Histone H3 N-Terminal Tail Revealed by High Precision Single-Molecule FRET

Synthesis of a Wheat/Maize Hybrid CENH3 Gene, the Genetic Transformation of Wheat, Its Chromosomal Localization and Effects on Chromosome Behaviors in Wheat/Maize Somatic Hybrids

Centromere-specific histone H3 (CENH3) replaces the canonical histone H3 in nucleosomes of functional centromeres, and plays important roles in faithful chromosome segregation during cell division. CENH3 is also important in the recognition of alien centromeres and determines the accommodation or elimination of alien chromosomes in interspecific or intergenic hybridization. In this study, a maize full length CENH3 with a yellow fluorescent protein (YFP) tag at C-terminus (ZmCENH3-YFP) and a synthetic hybrid wmCENH3 with the N-terminus from wheat CENH3 and the histone fold domain (HFD) from maize tagged with a red fluorescent protein (RFP) at the C-terminus (wmCENH3-RFP) were transformed to wheat by biolistics transformation. Transgenic wheat plants with both ZmCNEH3-YFP and wmCENH3-RFP genes were identified by PCR. The expression of ZmCENH3-YFP was not observed, while the expression of wmCENH3-RFP could be detected by RT-PCR, direct fluorescence microscopy, and immunostaining with anti-RFP antibody. The expressed wmCENH3-RFP was localized to nuclei as dotted patterns, indicating its targeting to wheat centromeres. Somatic hybridization was performed between wmCENH3-RFP transgenic wheat and transgenic maize that expressed a ZmCENH3-YFP gene to investigate chromosome behaviors in somatic hybrids. Cytological and FISH analyses of somatic hybrid cells showed the formation of micronuclei and lagging chromatin in both somatic hybridizations with or without the wmCENH3-RFP transgene, indicating that ectopically expressed wmCENH3 could not overcome chromosome elimination in wheat/maize somatic hybrids. Immunostaining of wmCENH3-RFP and ZmCENH3-YFP in early stage somatic hybrid cells indicated that both wmCENH3-RFP and ZmCENH3-YFP proteins were expressed, but their binding patterns changed from the commonly observed dotted patterns to diffused ones, suggesting that the inactivation of CENH3 might be a factor for chromosome elimination in wheat/maize somatic hybridization.

Can Reprogramming of Overall Epigenetic Memory and Specific Parental Genomic Imprinting Memory within Donor Cell-Inherited Nuclear Genome be a Major Hindrance for the Somatic Cell Cloning of Mammals? – A Review

Abstract Successful cloning of animals by somatic cell nuclear transfer (SCNT) requires epigenetic transcriptional reprogramming of the differentiated state of the donor cell nucleus to a totipotent embryonic ground state. It means that the donor nuclei must cease its own program of gene expression and restore a particular program of the embryonic genome expression regulation that is necessary for normal development. Transcriptional activity of somatic cell-derived nuclear genome during embryo pre- and postimplantation development as well as foetogenesis is correlated with the frequencies for spatial remodeling of chromatin architecture and reprogramming of cellular epigenetic memory. This former and this latter process include such covalent modifications as demethylation/re-methylation of DNA cytosine residues and acetylation/deacetylation as well as demethylation/re-methylation of lysine residues of nucleosomal core-derived histones H3 and H4. The main cause of low SCNT efficiency in mammals turns out to be an incomplete reprogramming of transcriptional activity for donor cell-descended genes. It has been ascertained that somatic cell nuclei should undergo the wide DNA cytosine residue demethylation changes throughout the early development of cloned embryos to reset their own overall epigenetic and parental genomic imprinting memories that have been established by re-methylation of the nuclear donor cell-inherited genome during specific pathways of somatic and germ cell lineage differentiation. A more extensive understanding of the molecular mechanisms and recognition of determinants for epigenetic transcriptional reprogrammability of somatic cell nuclear genome will be helpful to solve the problems resulting from unsatisfactory SCNT effectiveness and open new possibilities for common application of this technology in transgenic research focused on human biomedicine.

Skin vaccination using microneedles coated with a plasmid DNA cocktail encoding nucleosomal histones of Leishmania spp.

Vaccine delivery using microneedles (MNs) represents a safe, easily disposable and painless alternative to traditional needle immunizations. The MN delivery of DNA vaccines to the dermis may result in a superior immune response and/or an equivalent immune response at a lower vaccine dose (dose-sparing). This could be of special interest for immunization programs against neglected tropical diseases such as leishmaniasis. In this work, we loaded a MN device with 60μg of a plasmid DNA cocktail encoding the Leishmania infantum nucleosomal histones H2A, H2B, H3 and H4 and compared its immunogenicity and protective capacity against conventional s.c. or i.d. injection of the plasmid. Mice immunized with MNs showed increased ratios of IFN-γ/IL-10, IFN-γ/IL-13, IFN-γ/IL-4, and IFN-γ/TGF-β in the spleens and lymph nodes compared with mice immunized by s.c. and i.d. routes. Furthermore, CCXCL9, CXCL10 and CCL2 levels were also higher. These data suggest that the nucleic acid immunization using MNs produced a better bias towards a Th1 response. However, none of the immunizations strategies were able to control Leishmania major infection in BALB/c mice, as illustrated by an increase in lesion size and parasite burden.

The NuA4 Core Complex Acetylates Nucleosomal Histone H4 through a Double Recognition Mechanism

NuA4 catalyzes the acetylation of nucleosomes at histone H4, which is a well-established epigenetic event, controlling many genomic processes in Saccharomyces cerevisiae. Here we report the crystal structures of the NuA4 core complex and a cryoelectron microscopy structure with the nucleosome. The structures show that the histone-binding pocket of the enzyme is rearranged, suggesting its activation. The enzyme binds the histone tail mainly through the target lysine residue, with a preference for a small residue at the -1 position. The complex engages the nucleosome at the dish face and orients its catalytic pocket close to the H4 tail to achieve selective acetylation. The combined data reveal a space-sequence double recognition mechanism of the histone tails by a modifying enzyme in the context of the nucleosome.

Differentially Isotope-Labeled Nucleosomes To Study Asymmetric Histone Modification Crosstalk by Time-Resolved NMR Spectroscopy.

Post-translational modifications (PTMs) of histones regulate chromatin structure and function. Because nucleosomes contain two copies each of the four core histones, the establishment of different PTMs on individual "sister" histones in the same nucleosomal context, that is, asymmetric histone PTMs, are difficult to analyze. Here, we generated differentially isotope-labeled nucleosomes to study asymmetric histone modification crosstalk by time-resolved NMR spectroscopy. Specifically, we present mechanistic insights into nucleosomal histone H3 modification reactions in cis and in trans, that is, within individual H3 copies or between them. We validated our approach by using the H3S10phK14ac crosstalk mechanism, which is mediated by the Gcn5 acetyltransferase. Moreover, phosphorylation assays on methylated substrates showed that, under certain conditions, Haspin kinase is able to produce nucleosomes decorated asymmetrically with two distinct types of PTMs.

Differentially Isotope‐Labeled Nucleosomes To Study Asymmetric Histone Modification Crosstalk by Time‐Resolved NMR Spectroscopy

AbstractPost‐translational modifications (PTMs) of histones regulate chromatin structure and function. Because nucleosomes contain two copies each of the four core histones, the establishment of different PTMs on individual “sister” histones in the same nucleosomal context, that is, asymmetric histone PTMs, are difficult to analyze. Here, we generated differentially isotope‐labeled nucleosomes to study asymmetric histone modification crosstalk by time‐resolved NMR spectroscopy. Specifically, we present mechanistic insights into nucleosomal histone H3 modification reactions in cis and in trans, that is, within individual H3 copies or between them. We validated our approach by using the H3S10phK14ac crosstalk mechanism, which is mediated by the Gcn5 acetyltransferase. Moreover, phosphorylation assays on methylated substrates showed that, under certain conditions, Haspin kinase is able to produce nucleosomes decorated asymmetrically with two distinct types of PTMs.

RNF 4 interacts with both SUMO and nucleosomes to promote the DNA damage response

The post-translational modification of DNA repair and checkpoint proteins by ubiquitin and small ubiquitin-like modifier (SUMO) critically orchestrates the DNA damage response (DDR). The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin, through its selective recognition and ubiquitination of SUMO-modified proteins. Here, we define a key new determinant for target discrimination by RNF4, in addition to interaction with SUMO. We identify a nucleosome-targeting motif within the RNF4 RING domain that can bind DNA and thereby enables RNF4 to selectively ubiquitinate nucleosomal histones. Furthermore, RNF4 nucleosome-targeting is crucially required for the repair of TRF2-depleted dysfunctional telomeres by 53BP1-mediated non-homologous end joining.

The N-terminus and Tudor domains of Sgf29 are important for its heterochromatin boundary formation function

Eukaryotic chromosomes are organized into heterochromatin and euchromatin domains. Heterochromatin domains are transcriptionally repressed and prevented from spreading into neighbouring genes by chromatin boundaries. Previously, we identified 55 boundary-related genes in Saccharomyces cerevisiae. In this study, we describe the characterization of one of these boundary genes, named SGF29, which was previously reported as a component of the SAGA, SLIK, ADA and HAT-A2 complex. A domain analysis of Sgf29 identified two minimal regions that can function as individual boundaries. The N-terminal minimal region comprising amino acids 1-12, which has not been defined as a functional domain, showed stronger boundary formation ability than the C-terminal minimal region comprising amino acids 110-255, which contains Tudor domains. Together with Ada2, Ada3 and Sgf29, which are all components of SAGA, Gcn5 acetylates multiple lysine residues on nucleosomal histone H3, which is associated with an open chromatin structure. However, the results presented in this study suggest that the boundary formation ability of the Sgf29 minimal regions is independent of Gcn5. An in vivo analysis also revealed that Sgf29 and Gcn5 perform distinct functions at native telomere boundary regions on the chromosome.

Phospho-dependent recruitment of NuA4 by MRX at DNA breaks regulates RPA dynamics during resection

Background The NuA4 histone acetyltransferase is a highly conserved multisubunit complex responsible for acetylation of nucleosomal histone H4 and H2A. Mutations in different NuA4 subunits create cell cycle arrest or delay in G2/M. The NuA4 complex is important for gene expression but also the efficient repair of DNA double strand breaks (DSBs) in vivo. NuA4 is rapidly recruited to chromatin surrounding a DSB in vivo at the same time histone H2A (X) is phosphorylated in the neighboring region (g-H2AX). While we have shown that NuA4 can interact with phosphorylated H2A(X), we speculated that another interaction was required for the initial recruitment of NuA4 at the break. Based on the presence of an ATM-related factor within NuA4, the subunitTra1 (TRRAP), it was tempting to postulate that DSB sensing factors known to recruit ATM family PIKK factors could be responsible for NuA4 recruitment.

CENP-A Nucleosomes adopt an Unconventional Shape

The centromere is the locus that ensures proper segregation of chromosomes through generations. In all eukaryotes, except for budding yeast, centromere location is maintained by an epigenetic mechanism. The most attractive candidate to provide the centromere-specifying epigenetic mark is the histone variant centromere protein A (CENP-A) that replaces histone H3 in centromeric nucleosomes. The (CENP-A/H4)2 heterotetramer crystal structure (Sekulic et al., 2010, Nature 467:347-351) led to our proposal that CENP-A could distinguish centromeric chromatin from the rest of the chromosome via structural deviation from within the folded octameric core of the nucleosome. Our model is based on the intranucleosomal rigidity conferred by CENP-A and rotation at the CENP-A/CENP-A interface. If preserved upon nucleosome formation the rotation would alter the radius of curvature of nucleosomal DNA and histone subunit packing. In the subsequent crystal structure of the CENP-A nucleosome (Tachiwana et al., 2011, Nature 476:232-235), though, nucleosomal DNA curvature and histone subunit packing are as in conventional nucleosome. To investigate the conformation of CENP-A nucleosomes in solution we used intranucleosomal FRET. We find that CENP-A nucleosomes heavily populate an atypical conformation where H2A/H2B dimers are 4 Å further apart at steady state relative to conventional nucleosomes. These data suggest that CENP-A nucleosomes in solution exist in equilibrium between at least two structural conformations: one that is similar to the conventional nucleosomes and one that is altered by rotation at the CENP-A/CENP-A interface. To further interrogate the structure of CENP-A nucleosomes in solution we are also employing SAXS (small-angle X-ray scattering) and SANS (small-angle neutron scattering). SANS experiments exploit contrast variation schemes to provide information on DNA and protein subunits independently. Our ongoing biophysical studies promise to provide critical insight into how CENP-A nucleosomes distinguish centromeric chromatin in the context of mammalian chromosomes.

Hinge and Chromoshadow of HP1α Participate in Recognition of K9 Methylated Histone H3 in Nucleosomes

The majority of the genome in eukaryotes is packaged into transcriptionally inactive chromatin. Heterochromatin protein 1 (HP1) is a major player in the establishment and maintenance of heterochromatin. HP1 specifically recognizes a methylated lysine residue at position 9 in histone H3 through its N-terminal chromo domain (CD). To elucidate the binding properties of HP1α to nucleosomes in vitro, we reconstituted nucleosomes containing histone H3 trimethylated at lysine 9. HP1α exhibited high-affinity binding to nucleosomes containing methylated histone H3 in a nucleosome core-number-dependent manner. The hinge region (HR) connecting the CD and C-terminal chromoshadow domain (CSD), and the CSD contributed to the selective binding of HP1α to histone H3 with trimethylated lysine 9 through weak DNA binding and by suppressing the DNA binding, respectively. We propose that not only the specific recognition of lysine 9 methylation of histone H3 by the CD but also the HR and the CSD cooperatively contribute to the selective binding of HP1α to histone H3 lysine 9 methylated nucleosomes.