RESUMO
The human Mixed Lineage Leukemia-1 (MLL1) complex methylates histone H3K4 to promote transcription and is stimulated by monoubiquitination of histone H2B. Recent structures of the MLL1-WRAD core complex, which comprises the MLL1 methyltransferase, WDR5, RbBp5, Ash2L, and DPY-30, have revealed variability in the docking of MLL1-WRAD on nucleosomes. In addition, portions of the Ash2L structure and the position of DPY30 remain ambiguous. We used an integrated approach combining cryoelectron microscopy (cryo-EM) and mass spectrometry cross-linking to determine a structure of the MLL1-WRAD complex bound to ubiquitinated nucleosomes. The resulting model contains the Ash2L intrinsically disordered region (IDR), SPRY insertion region, Sdc1-DPY30 interacting region (SDI-motif), and the DPY30 dimer. We also resolved three additional states of MLL1-WRAD lacking one or more subunits, which may reflect different steps in the assembly of MLL1-WRAD. The docking of subunits in all four states differs from structures of MLL1-WRAD bound to unmodified nucleosomes, suggesting that H2B-ubiquitin favors assembly of the active complex. Our results provide a more complete picture of MLL1-WRAD and the role of ubiquitin in promoting formation of the active methyltransferase complex.
Assuntos
Histona-Lisina N-Metiltransferase , Peptídeos e Proteínas de Sinalização Intracelular , Proteína de Leucina Linfoide-Mieloide , Nucleossomos , Ubiquitinação , Microscopia Crioeletrônica , Histona-Lisina N-Metiltransferase/química , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteína de Leucina Linfoide-Mieloide/química , Proteína de Leucina Linfoide-Mieloide/genética , Nucleossomos/enzimologia , Ligação ProteicaRESUMO
The antigenic variation is an essential mechanism employed by the malaria parasite to establish a chronic infection in humans. Three major virulent proteins EMP1, RIFINs, and STEVOR have been implicated in contributing to the antigenic variation process and are encoded by multigene families in Plasmodium spp. The key virulence factor PfEMP1 is encoded by var genes, and it exhibits a mutually exclusive transcriptional switching between var genes, ensuring an individual parasite only transcribes a single var gene at a time. Expression of var genes is tightly regulated by two histone epigenetic methylation marks H3K36me3 and H3K9me3, of which the H3K36me3 mark is highly enriched on transcription start sites (TSSs) of suppressed var genes in P. falciparum. However, the mechanisms of H3K36me3 mark propagation on all the 59 var genes of P. falciparum are not known. Here, we have identified a PHD (Plant Homeodomain-like Domain) like domain present within the PfSET2 protein that specifically binds to the H3K36me2 mark, an intermediate product of the H3K36me3 mark formation on the nucleosome. Surprisingly, we have found that PHD - H3K36me2 interaction leads to stimulation of SET2 domain activity on the nucleosome substrates. The allosteric stimulation of the PfSET2 domain by PHD-like domain present within the same protein suggests a novel mechanism of H3K36me3 mark propagation on var genes of P. falciparum. This study proposes allosteric regulation of PfSET2 protein by H3K36me2 mark as an essential mechanism of var genes suppression to ensure successful antigenic variation by the malaria parasite.
Assuntos
Código das Histonas , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Regulação Alostérica , Metilação , Nucleossomos/enzimologia , Domínios ProteicosRESUMO
Dot1 (disruptor of telomeric silencing-1), the histone H3 lysine 79 (H3K79) methyltransferase, is conserved throughout evolution, and its deregulation is found in human leukemias. Here, we provide evidence that acetylation of histone H4 allosterically stimulates yeast Dot1 in a manner distinct from but coordinating with histone H2B ubiquitination (H2BUb). We further demonstrate that this stimulatory effect is specific to acetylation of lysine 16 (H4K16ac), a modification central to chromatin structure. We provide a mechanism of this histone cross-talk and show that H4K16ac and H2BUb play crucial roles in H3K79 di- and trimethylation in vitro and in vivo. These data reveal mechanisms that control H3K79 methylation and demonstrate how H4K16ac, H3K79me, and H2BUb function together to regulate gene transcription and gene silencing to ensure optimal maintenance and propagation of an epigenetic state.
Assuntos
Montagem e Desmontagem da Cromatina , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilação , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Nucleossomos/enzimologia , Conformação Proteica , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
DNA methylation and histone tail modifications are interrelated mechanisms involved in a wide range of biological processes, and disruption of this crosstalk is linked to diseases such as acute myeloid leukemia. In addition, DNA methyltransferase 3A (DNMT3A) activity is modulated by several regulatory proteins, including p53 and thymine DNA glycosylase (TDG). However, the relative role of histone tails and regulatory proteins in the simultaneous coordination of DNMT3A activity remains obscure. We observed that DNMT3A binds H3 tails and p53 or TDG at distinct allosteric sites to form DNMT3A-H3 tail-p53 or -TDG multiprotein complexes. Functional characterization of DNMT3A-H3 tail-p53 or -TDG complexes on human-derived synthetic histone H3 tails, mononucleosomes, or polynucleosomes shows p53 and TDG play dominant roles in the modulation of DNMT3A activity. Intriguingly, this dominance occurs even when DNMT3A is actively methylating nucleosome substrates. The activity of histone modifiers is influenced by their ability to sense modifications on histone tails within the same nucleosome or histone tails on neighboring nucleosomes. In contrast, we show here that DNMT3A acts on DNA within a single nucleosome, on nucleosomal DNA within adjacent nucleosomes, and DNA not associated with the DNMT3A-nucleosome complex. Our findings have direct bearing on how the histone code drives changes in DNA methylation and highlight the complex interplay between histone tails, epigenetic enzymes, and modulators of enzymatic activity.
Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Nucleossomos/enzimologia , Timina DNA Glicosilase/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Sítio Alostérico , DNA/metabolismo , Metilação de DNA , DNA Metiltransferase 3A , Epigênese Genética , Histonas/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Processamento de Proteína Pós-Traducional , Especificidade por SubstratoRESUMO
Cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) synthase (cGAS) recognizes cytosolic foreign or damaged DNA to activate the innate immune response to infection, inflammatory diseases, and cancer. By contrast, cGAS reactivity against self-DNA in the nucleus is suppressed by chromatin tethering. We report a 3.3-angstrom-resolution cryo-electron microscopy structure of cGAS in complex with the nucleosome core particle. The structure reveals that cGAS uses two conserved arginines to anchor to the nucleosome acidic patch. The nucleosome-binding interface exclusively occupies the strong double-stranded DNA (dsDNA)-binding surface on cGAS and sterically prevents cGAS from oligomerizing into the functionally active 2:2 cGAS-dsDNA state. These findings provide a structural basis for how cGAS maintains an inhibited state in the nucleus and further exemplify the role of the nucleosome in regulating diverse nuclear protein functions.
Assuntos
Proteínas Nucleares/química , Nucleossomos/enzimologia , Nucleotidiltransferases/química , Domínio Catalítico , Microscopia Crioeletrônica , DNA/química , Humanos , Multimerização ProteicaRESUMO
The cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) senses invasion of pathogenic DNA and stimulates inflammatory signaling, autophagy, and apoptosis. Organization of host DNA into nucleosomes was proposed to limit cGAS autoinduction, but the underlying mechanism was unknown. Here, we report the structural basis for this inhibition. In the cryo-electron microscopy structure of the human cGAS-nucleosome core particle (NCP) complex, two cGAS monomers bridge two NCPs by binding the acidic patch of the histone H2A-H2B dimer and nucleosomal DNA. In this configuration, all three known cGAS DNA binding sites, required for cGAS activation, are repurposed or become inaccessible, and cGAS dimerization, another prerequisite for activation, is inhibited. Mutating key residues linking cGAS and the acidic patch alleviates nucleosomal inhibition. This study establishes a structural framework for why cGAS is silenced on chromatinized self-DNA.
Assuntos
Proteínas Nucleares/química , Nucleossomos/enzimologia , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Animais , Domínio Catalítico , Microscopia Crioeletrônica , DNA/química , Humanos , Nucleotidiltransferases/genética , Conformação Proteica , XenopusRESUMO
In mammalian cells, distinct H3K4 methylation states are created by deposition of methyl groups by multiple complexes of histone lysine methyltransferase 2 (KMT2) family proteins. For comprehensive analyses that directly compare the catalytic properties of all six human KMT2 complexes, we employed a biochemically defined system reconstituted with recombinant KMT2 core complexes (KMT2CoreCs) containing minimal components required for nucleosomal H3K4 methylation activity. We found that each KMT2CoreC generates distinct states and different levels of H3K4 methylation, and except for MLL3 all are stimulated by H2Bub. Notably, SET1BCoreC exhibited the strongest H3K4 methylation activity and, to our surprise, did not require H2B ubiquitylation (H2Bub); in contrast, H2Bub was required for the H3K4me2/3 activity of the paralog SET1ACoreC. We also found that WDR5, RbBP5, ASH2L and DPY30 are required for efficient H3K4 methyltransferase activities of all KMT2CoreCs except MLL3, which could produce H3K4me1 in the absence of WDR5. Importantly, deletion of the PHD2 domain of CFP1 led to complete loss of the H3K4me2/3 activities of SET1A/BCoreCs in the presence of H2Bub, indicating a critical role for this domain in the H2Bub-stimulated H3K4 methylation. Collectively, our results suggest that each KMT2 complex methylates H3K4 through distinct mechanisms in which individual subunits differentially participate.
Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Ubiquitinação , Proteínas de Ligação a DNA/metabolismo , Histona-Lisina N-Metiltransferase/química , Humanos , Metilação , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Neoplasias/metabolismo , Nucleossomos/enzimologia , Domínios Proteicos , Subunidades Proteicas/metabolismoRESUMO
Among the widespread and increasing number of identified post-translational modifications (PTMs), arginine methylation is catalyzed by the protein arginine methyltransferases (PRMTs) and regulates fundamental processes in cells, such as gene regulation, RNA processing, translation, and signal transduction. As epigenetic regulators, PRMTs play key roles in pluripotency, differentiation, proliferation, survival, and apoptosis, which are essential biological programs leading to development, adult homeostasis but also pathological conditions including cancer. A full understanding of the molecular mechanisms that underlie PRMT-mediated gene regulation requires the genome wide mapping of each player, i.e., PRMTs, their substrates and epigenetic marks, methyl-marks readers as well as interaction partners, in a thorough and unambiguous manner. However, despite the tremendous advances in high throughput sequencing technologies and the numerous efforts from the scientific community, the epigenomic profiling of PRMTs as well as their histone and non-histone substrates still remains a big challenge owing to obvious limitations in tools and methodologies. This review will summarize the present knowledge about the genome wide mapping of PRMTs and their substrates as well as the technical approaches currently in use. The limitations and pitfalls of the technical tools along with conventional approaches will be then discussed in detail. Finally, potential new strategies for chromatin profiling of PRMTs and histone substrates will be proposed and described.
Assuntos
Imunoprecipitação da Cromatina/métodos , Epigenoma , Epigenômica/métodos , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/metabolismo , Animais , Arginina/metabolismo , Cromatina/enzimologia , Cromatina/metabolismo , Inibidores Enzimáticos/química , Histonas/química , Humanos , Metilação , Mutação , Nucleossomos/enzimologia , Nucleossomos/metabolismo , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/genéticaRESUMO
Nucleosome positioning controls the accessible regions of chromatin and plays essential roles in DNA-templated processes. ATP driven remodeling enzymes are known to be crucial for its establishment in vivo, but their nonequilibrium nature has hindered the development of a unified theoretical framework for nucleosome positioning. Using a perturbation theory, we show that the effect of these enzymes can be well approximated by effective equilibrium models with rescaled temperatures and interactions. Numerical simulations support the accuracy of the theory in predicting both kinetic and steady-state quantities, including the effective temperature and the radial distribution function, in biologically relevant regimes. The energy landscape view emerging from our study provides an intuitive understanding for the impact of remodeling enzymes in either reinforcing or overwriting intrinsic signals for nucleosome positioning, and may help improve the accuracy of computational models for its prediction in silico.
Assuntos
Cromatina/metabolismo , Modelos Biológicos , Trifosfato de Adenosina/metabolismo , Cromatina/enzimologia , Cromatina/genética , DNA/genética , DNA/metabolismo , Humanos , Nucleossomos/enzimologia , Nucleossomos/genética , Nucleossomos/metabolismoRESUMO
DNA N6-adenine methylation (6mA) has recently been described in diverse eukaryotes, spanning unicellular organisms to metazoa. Here, we report a DNA 6mA methyltransferase complex in ciliates, termed MTA1c. It consists of two MT-A70 proteins and two homeobox-like DNA-binding proteins and specifically methylates dsDNA. Disruption of the catalytic subunit, MTA1, in the ciliate Oxytricha leads to genome-wide loss of 6mA and abolishment of the consensus ApT dimethylated motif. Mutants fail to complete the sexual cycle, which normally coincides with peak MTA1 expression. We investigate the impact of 6mA on nucleosome occupancy in vitro by reconstructing complete, full-length Oxytricha chromosomes harboring 6mA in native or ectopic positions. We show that 6mA directly disfavors nucleosomes in vitro in a local, quantitative manner, independent of DNA sequence. Furthermore, the chromatin remodeler ACF can overcome this effect. Our study identifies a diverged DNA N6-adenine methyltransferase and defines the role of 6mA in chromatin organization.
Assuntos
Complexos Multienzimáticos/metabolismo , Nucleossomos/enzimologia , Oxytricha/enzimologia , Proteínas de Protozoários/metabolismo , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Tetrahymena thermophila/enzimologia , Complexos Multienzimáticos/genética , Nucleossomos/genética , Oxytricha/genética , Proteínas de Protozoários/genética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética , Tetrahymena thermophila/genéticaRESUMO
Coordinated changes of DNA (de)methylation, nucleosome positioning, and chromatin binding of the architectural protein CTCF play an important role for establishing cell-type-specific chromatin states during differentiation. To elucidate molecular mechanisms that link these processes, we studied the perturbed DNA modification landscape in mouse embryonic stem cells (ESCs) carrying a double knockout (DKO) of the Tet1 and Tet2 dioxygenases. These enzymes are responsible for the conversion of 5-methylcytosine (5mC) into its hydroxymethylated (5hmC), formylated (5fC), or carboxylated (5caC) forms. We determined changes in nucleosome positioning, CTCF binding, DNA methylation, and gene expression in DKO ESCs and developed biophysical models to predict differential CTCF binding. Methylation-sensitive nucleosome repositioning accounted for a significant portion of CTCF binding loss in DKO ESCs, whereas unmethylated and nucleosome-depleted CpG islands were enriched for CTCF sites that remained occupied. A number of CTCF sites also displayed direct correlations with the CpG modification state: CTCF was preferentially lost from sites that were marked with 5hmC in wild-type (WT) cells but not from 5fC-enriched sites. In addition, we found that some CTCF sites can act as bifurcation points defining the differential methylation landscape. CTCF loss from such sites, for example, at promoters, boundaries of chromatin loops, and topologically associated domains (TADs), was correlated with DNA methylation/demethylation spreading and can be linked to down-regulation of neighboring genes. Our results reveal a hierarchical interplay between cytosine modifications, nucleosome positions, and DNA sequence that determines differential CTCF binding and regulates gene expression.
Assuntos
Metilação de DNA , Proteínas de Ligação a DNA/genética , Epigênese Genética , Células-Tronco Embrionárias Murinas/enzimologia , Proteínas Proto-Oncogênicas/genética , 5-Metilcitosina/química , Animais , Fator de Ligação a CCCTC/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Elementos Isolantes/genética , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/metabolismo , Nucleossomos/enzimologia , Proteínas Proto-Oncogênicas/metabolismoRESUMO
LSD1 and LSD2 are homologous histone demethylases with opposite biological outcomes related to chromatin silencing and transcription elongation, respectively. Unlike LSD1, LSD2 nucleosome-demethylase activity relies on a specific linker peptide from the multidomain protein NPAC. We used single-particle cryoelectron microscopy (cryo-EM), in combination with kinetic and mutational analysis, to analyze the mechanisms underlying the function of the human LSD2/NPAC-linker/nucleosome complex. Weak interactions between LSD2 and DNA enable multiple binding modes for the association of the demethylase to the nucleosome. The demethylase thereby captures mono- and dimethyl Lys4 of the H3 tail to afford histone demethylation. Our studies also establish that the dehydrogenase domain of NPAC serves as a catalytically inert oligomerization module. While LSD1/CoREST forms a nucleosome docking platform at silenced gene promoters, LSD2/NPAC is a multifunctional enzyme complex with flexible linkers, tailored for rapid chromatin modification, in conjunction with the advance of the RNA polymerase on actively transcribed genes.
Assuntos
Histona Desmetilases/metabolismo , Proteínas Nucleares/metabolismo , Nucleossomos/metabolismo , Oxirredutases/metabolismo , Sequência de Aminoácidos , Desmetilação , Histona Desmetilases/química , Histona Desmetilases/genética , Histonas/metabolismo , Humanos , Modelos Moleculares , Enzimas Multifuncionais/química , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Nucleossomos/enzimologia , Nucleossomos/genética , Oxirredutases/química , Oxirredutases/genética , Domínios ProteicosRESUMO
The vast majority of oxidized bases that form in DNA are subject to base excision repair (BER). The DNA intermediates generated during successive steps in BER may prove mutagenic or lethal, making it critical that they be 'handed' from one BER enzyme to the next in a coordinated fashion. Here, we report that the handoff of BER intermediates that occurs during the repair of naked DNA substrates differs significantly from that in nucleosomes. During BER of oxidized bases in naked DNA, products generated by the DNA glycosylase NTHL1 were efficiently processed by the downstream enzyme, AP-endonuclease (APE1). In nucleosomes, however, NTHL1-generated products accumulated to significant levels and persisted for some time. During BER of naked DNA substrates, APE1 completely bypasses the inefficient lyase activity of NTHL1. In nucleosomes, the NTHL1-associated lyase contributes to BER, even in the presence of APE1. Moreover, in nucleosomes but not in naked DNA, APE1 was able to process NTHL1 lyase-generated substrates just as efficiently as it processed abasic sites. Thus, the lyase activity of hNTHL1, and the 3' diesterase activity of APE1, which had been seen as relatively dispensable, may have been preserved during evolution to enhance BER in chromatin.
Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA/genética , Desoxirribonuclease (Dímero de Pirimidina)/genética , Nucleossomos/enzimologia , Cromatina/enzimologia , Cromatina/genética , DNA/química , Dano ao DNA/genética , DNA Glicosilases/química , DNA Glicosilases/genética , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , Desoxirribonuclease (Dímero de Pirimidina)/química , Esterases/genética , Humanos , Liases/química , Liases/genética , Nucleossomos/genética , OxirreduçãoRESUMO
DNA methylation and H3K9me are hallmarks of heterochromatin in plants and mammals, and are successfully maintained across generations. The biochemical and structural basis for this maintenance is poorly understood. The maintenance DNA methyltransferase from Zea mays, ZMET2, recognizes dimethylation of H3K9 via a chromodomain (CD) and a bromo adjacent homology (BAH) domain, which flank the catalytic domain. Here, we show that dinucleosomes are the preferred ZMET2 substrate, with DNA methylation preferentially targeted to linker DNA. Electron microscopy shows one ZMET2 molecule bridging two nucleosomes within a dinucleosome. We find that the CD stabilizes binding, whereas the BAH domain enables allosteric activation by the H3K9me mark. ZMET2 further couples recognition of H3K9me to an increase in the specificity for hemimethylated versus unmethylated DNA. We propose a model in which synergistic coupling between recognition of nucleosome spacing, H3K9 methylation, and DNA modification allows ZMET2 to maintain DNA methylation in heterochromatin with high fidelity.
Assuntos
Metilação de DNA , Metilases de Modificação do DNA/metabolismo , Nucleossomos/enzimologia , Proteínas de Plantas/metabolismo , Animais , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/ultraestrutura , Ativação Enzimática , Escherichia coli/enzimologia , Escherichia coli/genética , Microscopia Eletrônica , Modelos Moleculares , Conformação de Ácido Nucleico , Nucleossomos/química , Nucleossomos/genética , Nucleossomos/ultraestrutura , Proteínas de Plantas/genética , Proteínas de Plantas/ultraestrutura , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Especificidade por Substrato , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
The classic view of nucleosome organization at active promoters is that two well-positioned nucleosomes flank a nucleosome-depleted region (NDR). However, this view has been recently disputed by contradictory reports as to whether wider (â³150 bp) NDRs instead contain unstable, micrococcal nuclease-sensitive ("fragile") nucleosomal particles. To determine the composition of fragile particles, we introduce CUT&RUN.ChIP, in which targeted nuclease cleavage and release is followed by chromatin immunoprecipitation. We find that fragile particles represent the occupancy of the RSC (remodeling the structure of chromatin) nucleosome remodeling complex and RSC-bound, partially unwrapped nucleosomal intermediates. We also find that general regulatory factors (GRFs) bind to partially unwrapped nucleosomes at these promoters. We propose that RSC binding and its action cause nucleosomes to unravel, facilitate subsequent binding of GRFs, and constitute a dynamic cycle of nucleosome deposition and clearance at the subset of wide Pol II promoter NDRs.
Assuntos
Montagem e Desmontagem da Cromatina , Imunoprecipitação da Cromatina/métodos , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Nuclease do Micrococo/metabolismo , Nucleossomos/enzimologia , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Fatores de Transcrição/metabolismo , Sítios de Ligação , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Conformação de Ácido Nucleico , Nucleossomos/genética , Ligação Proteica , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genéticaRESUMO
Persistent DNA damage is responsible for mutagenesis, aging, and disease. Repair of the prototypic oxidatively damaged guanine lesion 8-oxo-7,8-dihydroguanine (8-oxoG) is initiated by oxoguanine glycosylase (hOGG1 in humans). In this work, we examine hOGG1 activity on DNA packaged as it is in chromatin, in a nucleosome core particle (NCP). We use synthetic methods to generate a population of NCPs with G to 8-oxoG substitutions and evaluate the global profile of hOGG1 repair in packaged DNA. For several turns of the helix, we observe that solution accessible 8-oxoGs are sites of activity for hOGG1. At the dyad axis, however, hOGG1 activity is suppressed, even at lesions predicted to be solution accessible by hydroxyl radical footprinting (HRF). We predict this diminished activity is due to the properties of the DNA unique to the dyad axis and/or the local histone environment. In contrast to the dyad axis, the DNA ends reveal hOGG1 activity at sites predicted by HRF to be both solution accessible and inaccessible. We attribute the lack of correlation between hOGG1 activity and solution accessibility at the ends of the DNA to transient unwrapping of the DNA from the protein core, thus exposing the inward-facing lesions.
Assuntos
DNA Glicosilases/metabolismo , DNA/metabolismo , Guanina/análogos & derivados , DNA/química , DNA Glicosilases/química , DNA Glicosilases/genética , Reparo do DNA , Guanina/química , Guanina/metabolismo , Humanos , Radical Hidroxila/química , Radical Hidroxila/metabolismo , Modelos Moleculares , Nucleossomos/química , Nucleossomos/enzimologia , Nucleossomos/metabolismo , Relação Estrutura-AtividadeRESUMO
PRDM9 is a zinc finger protein that binds DNA at specific locations in the genome where it trimethylates histone H3 at lysines 4 and 36 at surrounding nucleosomes. During meiosis in many species, including humans and mice where PRDM9 has been most intensely studied, these actions determine the location of recombination hotspots, where genetic recombination occurs. In addition, PRDM9 facilitates the association of hotspots with the chromosome axis, the site of the programmed DNA double-strand breaks (DSBs) that give rise to genetic exchange between chromosomes. In the absence of PRDM9 DSBs are not properly repaired. Collectively, these actions determine patterns of genetic linkage and the possibilities for chromosome reorganization over successive generations.
Assuntos
Genoma , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Meiose , Recombinação Genética , Animais , Quebras de DNA de Cadeia Dupla , Células Eucarióticas/citologia , Células Eucarióticas/enzimologia , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Metilação , Camundongos , Nucleossomos/enzimologia , Nucleossomos/genética , Domínios ProteicosRESUMO
BACKGROUND: Genome-wide association studies (GWASs) are used to identify genetic variants for association with bipolar disorder (BD) risk; however, each GWAS can only reveal a small fraction of this association. This study systematically analyzed multiple GWAS data sets to provide further insights into potential causal BD processes by integrating the results of Psychiatric Genomics Consortium Phase I (PGC-I) for BD with core human pathways and functional networks. METHODS: The i-Gsea4GwasV2 program was used to analyze data from the PGC-I GWAS for BD (the pathways came from Reactome), as well as the nominally significant pathways. We established a gene network of the significant pathways and performed a gene set analysis for each gene cluster of the Enhancing Neuroimaging Genetics through Meta-Analysis (ENIGMA) GWAS data for the volumes of the intracranial region and seven subcortical regions. RESULTS: A total of 30 of 1816 Reactome pathways were identified and showed associations with BD risk. We further revealed 22 interconnected functional and topologically interacting clusters (Clusters 0-21) that were associated with BD risk. Moreover, we obtained brain transcriptome data from BrainSpan and found significant associations between common variants of the genes in Cluster 1 with the hippocampus (HIP; P = .026; family-wise error [FWE] correction) and amygdala (AMY; P = .016; FEW correction) in Cluster 8 with HIP (P = .022; FWE correction). The genes in Cluster 1 were enriched for the transcriptional co-expression profile in the prenatal AMY, and core genes (CDH4, MTA2, RBBP4, and HDAC2) were identified to be involved in regulating early brain development. CONCLUSION: This study demonstrated that the HIP and AMY play a central role in neurodevelopment and BD risk.
Assuntos
Tonsila do Cerebelo , Transtorno Bipolar , Hipocampo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Nucleossomos/enzimologia , Transcriptoma/genética , Tonsila do Cerebelo/diagnóstico por imagem , Tonsila do Cerebelo/crescimento & desenvolvimento , Transtorno Bipolar/diagnóstico , Transtorno Bipolar/genética , Transtorno Bipolar/metabolismo , Predisposição Genética para Doença , Variação Genética , Estudo de Associação Genômica Ampla , Hipocampo/diagnóstico por imagem , Hipocampo/crescimento & desenvolvimento , Humanos , Neuroimagem/métodos , Transdução de Sinais/genéticaRESUMO
Human ALC1 is an oncogene-encoded chromatin-remodeling enzyme required for DNA repair that possesses a poly(ADP-ribose) (PAR)-binding macro domain. Its engagement with PARylated PARP1 activates ALC1 at sites of DNA damage, but the underlying mechanism remains unclear. Here, we establish a dual role for the macro domain in autoinhibition of ALC1 ATPase activity and coupling to nucleosome mobilization. In the absence of DNA damage, an inactive conformation of the ATPase is maintained by juxtaposition of the macro domain against predominantly the C-terminal ATPase lobe through conserved electrostatic interactions. Mutations within this interface displace the macro domain, constitutively activate the ALC1 ATPase independent of PARylated PARP1, and alter the dynamics of ALC1 recruitment at DNA damage sites. Upon DNA damage, binding of PARylated PARP1 by the macro domain induces a conformational change that relieves autoinhibitory interactions with the ATPase motor, which selectively activates ALC1 remodeling upon recruitment to sites of DNA damage.
Assuntos
Montagem e Desmontagem da Cromatina , Dano ao DNA , DNA Helicases/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Nucleossomos/enzimologia , Domínio Catalítico , Linhagem Celular Tumoral , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Humanos , Microscopia Eletrônica , Simulação de Dinâmica Molecular , Mutação , Nucleossomos/química , Poli(ADP-Ribose) Polimerase-1/química , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli ADP Ribosilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Espalhamento a Baixo Ângulo , Eletricidade Estática , Relação Estrutura-Atividade , Fatores de Tempo , Difração de Raios XRESUMO
Pituitary gonadotropin hormones are regulated by gonadotropin-releasing hormone (GnRH) via MAPK signaling pathways that stimulate gene transcription of the common α-subunit (Cga) and the hormone-specific ß-subunits of gonadotropin. We have reported previously that GnRH-induced activities at these genes include various histone modifications, but we did not examine histone phosphorylation. This modification adds a negative charge to residues of the histone tails that interact with the negatively charged DNA, is associated with closed chromatin during mitosis, but is increased at certain genes for transcriptional activation. Thus, the functions of this modification are unclear. We initially hypothesized that GnRH might induce phosphorylation of Ser-10 in histone 3 (H3S10p) as part of its regulation of gonadotropin gene expression, possibly involving cross-talk with H3K9 acetylation. We found that GnRH increases the levels of both modifications around the Cga gene transcriptional start site and that JNK inhibition dramatically reduces H3S10p levels. However, this modification had only a minor effect on Cga expression and no effect on H3K9ac. GnRH also increased H3S28p and H3K27ac levels and also those of activated mitogen- and stress-activated protein kinase 1 (MSK1). MSK1 inhibition dramatically reduced H3S28p levels in untreated and GnRH-treated cells and also affected H3K27ac levels. Although not affecting basal Cga expression, MSK1/2 inhibition repressed GnRH activation of Cga expression. Moreover, ChIP analysis revealed that GnRH-activated MSK1 targets the first nucleosome just downstream from the TSS. Given that the elongating RNA polymerase II (RNAPII) stalls at this well positioned nucleosome, GnRH-induced H3S28p, possibly in association with H3K27ac, would facilitate the progression of RNAPII.