RESUMEN
OBJECTIVE: In past work in budding yeast, we identified a nucleosomal region required for proper interactions between the histone chaperone complex yFACT and transcribed genes. Specific histone mutations within this region cause a shift in yFACT occupancy towards the 3' end of genes, a defect that we have attributed to impaired yFACT dissociation from DNA following transcription. In this work we wished to assess the contributions of DNA sequences at the 3' end of genes in promoting yFACT dissociation upon transcription termination. RESULTS: We generated fourteen different alleles of the constitutively expressed yeast gene PMA1, each lacking a distinct DNA fragment across its 3' end, and assessed their effects on occupancy of the yFACT component Spt16. Whereas most of these alleles conferred no defects on Spt16 occupancy, one did cause a modest increase in Spt16 binding at the gene's 3' end. Interestingly, the same allele also caused minor retention of RNA Polymerase II (Pol II) and altered nucleosome occupancy across the same region of the gene. These results suggest that specific DNA sequences at the 3' ends of genes can play roles in promoting efficient yFACT and Pol II dissociation from genes and can also contribute to proper chromatin architecture.
Asunto(s)
Nucleosomas , ARN Polimerasa II , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Nucleosomas/metabolismo , Nucleosomas/genética , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismo , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , ADN de Hongos/genética , ADN de Hongos/metabolismo , Alelos , Secuencia de Bases , Regulación Fúngica de la Expresión Génica , Transcripción GenéticaRESUMEN
Serial capture affinity purification (SCAP) is a powerful method to isolate a specific protein complex. When combined with cross-linking mass spectrometry and computational approaches, one can build an integrated structural model of the isolated complex. Here, we applied SCAP to dissect a subpopulation of WDR76 in complex with SPIN1, a histone reader that recognizes trimethylated histone H3 lysine4 (H3K4me3). In contrast to a previous SCAP analysis of the SPIN1:SPINDOC complex, histones and the H3K4me3 mark were enriched with the WDR76:SPIN1 complex. Next, interaction network analysis of copurifying proteins and microscopy analysis revealed a potential role of the WDR76:SPIN1 complex in the DNA damage response. Since we detected 149 pairs of cross-links between WDR76, SPIN1, and histones, we then built an integrated structural model of the complex where SPIN1 recognized the H3K4me3 epigenetic mark while interacting with WDR76. Finally, we used the powerful Bayesian Integrative Modeling approach as implemented in the Integrative Modeling Platform to build a model of WDR76 and SPIN1 bound to the nucleosome.
Asunto(s)
Daño del ADN , Histonas , Nucleosomas , Histonas/metabolismo , Histonas/química , Nucleosomas/metabolismo , Humanos , Unión Proteica , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas Portadoras/metabolismo , Proteínas Portadoras/química , Modelos Moleculares , ATPasas Asociadas con Actividades Celulares Diversas , ADN HelicasasRESUMEN
Linker histones play an essential role in chromatin packaging by facilitating compaction of the 11-nm fiber of nucleosomal "beads on a string." The result is a heterogeneous condensed state with local properties that range from dynamic, irregular, and liquid-like to stable and regular structures (the 30-nm fiber), which in turn impact chromatin-dependent activities at a fundamental level. The properties of the condensed state depend on the type of linker histone, particularly on the highly disordered C-terminal tail, which is the most variable region of the protein, both between species, and within the various subtypes and cell-type specific variants of a given organism. We have developed an in vitro model system comprising linker histone tail and linker DNA, which although very minimal, displays surprisingly complex behavior, and is sufficient to model the known states of linker histone-condensed chromatin: disordered "fuzzy" complexes ("open" chromatin), dense liquid-like assemblies (dynamic condensates), and higher-order structures (organized 30-nm fibers). A crucial advantage of such a simple model is that it allows the study of the various condensed states by NMR, circular dichroism, and scattering methods. Moreover, it allows capture of the thermodynamics underpinning the transitions between states through calorimetry. We have leveraged this to rationalize the distinct condensing properties of linker histone subtypes and variants across species that are encoded by the amino acid content of their C-terminal tails. Three properties emerge as key to defining the condensed state: charge density, lysine/arginine ratio, and proline-free regions, and we evaluate each separately using a strategic mutagenesis approach.
Asunto(s)
ADN , Histonas , Nucleosomas , Histonas/química , Histonas/metabolismo , Histonas/genética , ADN/química , ADN/metabolismo , Nucleosomas/metabolismo , Nucleosomas/química , Cromatina/química , Cromatina/metabolismo , Cromatina/genética , Animales , HumanosRESUMEN
During lagging strand chromatin replication, multiple Okazaki fragments (OFs) require processing and nucleosome assembly, but the mechanisms linking these processes remain unclear. Here, using transmission electron microscopy and rapid degradation of DNA ligase Cdc9, we observed flap structures accumulated on lagging strands, controlled by both Pol δ's strand displacement activity and Fen1's nuclease digestion. The distance between neighboring flap structures exhibits a regular pattern, indicative of matured OF length. While fen1Δ or enhanced strand displacement activities by polymerase δ (Pol δ; pol3exo-) minimally affect inter-flap distance, mutants affecting replication-coupled nucleosome assembly, such as cac1Δ and mcm2-3A, do significantly alter it. Deletion of Pol32, a subunit of DNA Pol δ, significantly increases this distance. Mechanistically, Pol32 binds to histone H3-H4 and is critical for nucleosome assembly on the lagging strand. Together, we propose that Pol32 establishes a connection between nucleosome assembly and the processing of OFs on lagging strands.
Asunto(s)
ADN Polimerasa III , ADN , Histonas , Nucleosomas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Nucleosomas/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , ADN Polimerasa III/metabolismo , ADN Polimerasa III/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , ADN/metabolismo , Replicación del ADN , Unión Proteica , ADN Polimerasa Dirigida por ADNRESUMEN
RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , ARN Polimerasa II , Transcripción Genética , Factores de Elongación Transcripcional , ARN Polimerasa II/metabolismo , ARN Polimerasa II/genética , Animales , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Factores de Elongación Transcripcional/metabolismo , Factores de Elongación Transcripcional/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Factor B de Elongación Transcripcional Positiva/metabolismo , Factor B de Elongación Transcripcional Positiva/genética , Regiones Promotoras Genéticas , Sistemas CRISPR-Cas , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Cromosomas Politénicos/genética , Cromosomas Politénicos/metabolismo , Regulación de la Expresión Génica , Fosforilación , Unión Proteica , Respuesta al Choque Térmico/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Nucleosomas/metabolismo , Nucleosomas/genéticaRESUMEN
In Saccharomyces cerevisiae, the forkhead (Fkh) transcription factor Fkh1 (forkhead homolog) enhances the activity of many DNA replication origins that act in early S-phase (early origins). Current models posit that Fkh1 acts directly to promote these origins' activity by binding to origin-adjacent Fkh1 binding sites (FKH sites). However, the post-DNA binding functions that Fkh1 uses to promote early origin activity are poorly understood. Fkh1 contains a conserved FHA (forkhead associated) domain, a protein-binding module with specificity for phosphothreonine (pT)-containing partner proteins. At a small subset of yeast origins, the Fkh1-FHA domain enhances the ORC (origin recognition complex)-origin binding step, the G1-phase event that initiates the origin cycle. However, the importance of the Fkh1-FHA domain to either chromosomal replication or ORC-origin interactions at genome scale is unclear. Here, S-phase SortSeq experiments were used to compare genome replication in proliferating FKH1 and fkh1-R80A mutant cells. The Fkh1-FHA domain promoted the activity of ≈ 100 origins that act in early to mid- S-phase, including the majority of centromere-associated origins, while simultaneously inhibiting ≈ 100 late origins. Thus, in the absence of a functional Fkh1-FHA domain, the temporal landscape of the yeast genome was flattened. Origins are associated with a positioned nucleosome array that frames a nucleosome depleted region (NDR) over the origin, and ORC-origin binding is necessary but not sufficient for this chromatin organization. To ask whether the Fkh1-FHA domain had an impact on this chromatin architecture at origins, ORC ChIPSeq data generated from proliferating cells and MNaseSeq data generated from G1-arrested and proliferating cell populations were assessed. Origin groups that were differentially regulated by the Fkh1-FHA domain were characterized by distinct effects of this domain on ORC-origin binding and G1-phase chromatin. Thus, the Fkh1-FHA domain controlled the distinct chromatin architecture at early origins in G1-phase and regulated origin activity in S-phase.
Asunto(s)
Cromatina , Replicación del ADN , Fase G1 , Complejo de Reconocimiento del Origen , Origen de Réplica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Origen de Réplica/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Replicación del ADN/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cromatina/genética , Cromatina/metabolismo , Complejo de Reconocimiento del Origen/genética , Complejo de Reconocimiento del Origen/metabolismo , Fase G1/genética , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Fase S/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Dominios Proteicos/genética , Sitios de Unión , Unión Proteica , Cromosomas Fúngicos/genética , Cromosomas Fúngicos/metabolismo , Nucleosomas/metabolismo , Nucleosomas/genéticaRESUMEN
Embryonic germ cells develop rapidly to establish the foundation for future developmental trajectories, and in this process, they make critical lineage choices including the configuration of their unique identity and a decision on sex. Here, we use single-cell genomics patterns for the entire embryonic germline in Drosophila melanogaster along with the somatic gonadal precursors after embryonic gonad coalescence to investigate molecular mechanisms involved in the setting up and regulation of the germline program. Profiling of the early germline chromatin landscape revealed sex- and stage-specific features. In the male germline immediately after zygotic activation, the chromatin structure underwent a brief remodeling phase during which nucleosome density was lower and deconcentrated from promoter regions. These findings echoed enrichment analysis results of our genomics data in which top candidates were factors with the ability to mediate large-scale chromatin reorganization. Together, they point to the importance of chromatin regulation in the early germline and raise the possibility of a conserved epigenetic reprogramming-like process required for proper initiation of germline development.
Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina , Drosophila melanogaster , Desarrollo Embrionario , Animales , Masculino , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Cromatina/metabolismo , Cromatina/genética , Ensamble y Desensamble de Cromatina/genética , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica/genética , Células Germinales Embrionarias/metabolismo , Células Germinales Embrionarias/citología , Células Germinativas/metabolismo , Epigénesis Genética , Femenino , Nucleosomas/metabolismo , Nucleosomas/genética , Análisis de la Célula Individual/métodosRESUMEN
Asymmetric cell divisions (ACDs) generate two daughter cells with identical genetic information but distinct cell fates through epigenetic mechanisms. However, the process of partitioning different epigenetic information into daughter cells remains unclear. Here, we demonstrate that the nucleosome remodeling and deacetylase (NuRD) complex is asymmetrically segregated into the surviving daughter cell rather than the apoptotic one during ACDs in Caenorhabditis elegans. The absence of NuRD triggers apoptosis via the EGL-1-CED-9-CED-4-CED-3 pathway, while an ectopic gain of NuRD enables apoptotic daughter cells to survive. We identify the vacuolar H+-adenosine triphosphatase (V-ATPase) complex as a crucial regulator of NuRD's asymmetric segregation. V-ATPase interacts with NuRD and is asymmetrically segregated into the surviving daughter cell. Inhibition of V-ATPase disrupts cytosolic pH asymmetry and NuRD asymmetry. We suggest that asymmetric segregation of V-ATPase may cause distinct acidification levels in the two daughter cells, enabling asymmetric epigenetic inheritance that specifies their respective life-versus-death fates.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , ATPasas de Translocación de Protón Vacuolares , Caenorhabditis elegans/genética , Animales , ATPasas de Translocación de Protón Vacuolares/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , División Celular Asimétrica , Apoptosis , Epigénesis Genética , Nucleosomas/metabolismoRESUMEN
The piRNA pathway is a conserved germline-specific small RNA pathway that ensures genomic integrity and continued fertility. In C. elegans and other nematodes, Type-I piRNAs are expressed from >10,000 independently transcribed genes clustered within two discrete domains of 1.5 and 3.5 MB on Chromosome IV. Clustering of piRNA genes contributes to their germline-specific expression, but the underlying mechanisms are unclear. We analyze isolated germ nuclei to demonstrate that the piRNA genomic domains are located in a heterochromatin-like environment. USTC (Upstream Sequence Transcription Complex) promotes strong association of nucleosomes throughout piRNA clusters, yet organizes the local nucleosome environment to direct the exposure of individual piRNA genes. Localization of USTC to the piRNA domains depends upon the ATPase chromatin remodeler ISW-1, which maintains high nucleosome density across piRNA clusters and ongoing production of piRNA precursors. Overall, this work provides insight into how chromatin states coordinate transcriptional regulation over large genomic domains, with implications for global genome organization.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Células Germinativas , Nucleosomas , Regiones Promotoras Genéticas , ARN Interferente Pequeño , Animales , Caenorhabditis elegans/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Nucleosomas/genética , Nucleosomas/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Células Germinativas/metabolismo , Ensamble y Desensamble de Cromatina/genética , Cromatina/genética , Cromatina/metabolismo , Transcripción Genética , Regulación de la Expresión Génica/genética , Heterocromatina/genética , Heterocromatina/metabolismo , ARN de Interacción con PiwiRESUMEN
Cells switch genes ON or OFF by altering the state of chromatin via histone modifications at specific regulatory locations along the chromatin polymer. These gene regulation processes are carried out by a network of reactions in which the histone marks spread to neighboring regions with the help of enzymes. In the literature, this spreading has been studied as a purely kinetic, non-diffusive process considering the interactions between neighboring nucleosomes. In this work, we go beyond this framework and study the spreading of modifications using a reaction-diffusion (RD) model accounting for the diffusion of the constituents. We quantitatively segregate the modification profiles generated from kinetic and RD models. The diffusion and degradation of enzymes set a natural length scale for limiting the domain size of modification spreading, and the resulting enzyme limitation is inherent in our model. We also demonstrate the emergence of confined modification domains without the explicit requirement of a nucleation site. We explore polymer compaction effects on spreading and show that single-cell domains may differ from averaged profiles. We find that the modification profiles from our model are comparable with existing H3K9me3 data of S. pombe.
Asunto(s)
Histonas , Histonas/metabolismo , Histonas/química , Difusión , Schizosaccharomyces/metabolismo , Schizosaccharomyces/genética , Nucleosomas/metabolismo , Nucleosomas/química , Código de Histonas , Cinética , Cromatina/metabolismo , Cromatina/química , Biología Computacional , Procesamiento Proteico-PostraduccionalRESUMEN
Clinical success with poly (ADP-ribose) polymerase inhibitors (PARPi) is impeded by inevitable resistance and associated cytotoxicity. Depletion of Amplified in Liver Cancer 1 (ALC1), a chromatin-remodeling enzyme, can overcome these limitations by hypersensitizing BReast CAncer genes 1/2 (BRCA1/2) mutant cells to PARPi. Here, we demonstrate that PARPi hypersensitivity upon ALC1 loss is reliant on its role in promoting the repair of chromatin buried abasic sites. We show that ALC1 enhances the ability of the abasic site processing enzyme, Apurinic/Apyrimidinic endonuclease 1 (APE1) to cleave nucleosome-occluded abasic sites. However, unrepaired abasic sites in ALC1-deficient cells are readily accessed by APE1 at the nucleosome-free replication forks. APE1 cleavage leads to fork breakage and trapping of PARP1/2 upon PARPi treatment, resulting in hypersensitivity. Collectively, our studies reveal how cells overcome the chromatin barrier to repair abasic lesions and uncover cleavage of abasic sites as a mechanism to overcome limitations of PARPi.
Asunto(s)
Proteína BRCA1 , Proteína BRCA2 , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Humanos , Línea Celular Tumoral , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/deficiencia , ADN-(Sitio Apurínico o Apirimidínico) Liasa/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/deficiencia , Reparación del ADN/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Femenino , Cromatina/metabolismo , Mutación , Daño del ADN/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Replicación del ADN/efectos de los fármacos , Nucleosomas/metabolismo , ADN Helicasas , Proteínas de Unión al ADNRESUMEN
Since the introduction of next generation sequencing technologies, the field of epigenomics has evolved rapidly. However, most commonly used assays are enrichment-based methods and thus only semi-quantitative. Nucleosome occupancy and methylome sequencing (NOMe-seq) allows for quantitative inference of chromatin states with single locus resolution, but this requires high sequencing depth and is therefore prohibitively expensive to routinely apply to organisms with large genomes. To overcome this limitation, we introduce guidedNOMe-seq, where we combine NOMe profiling with large scale sgRNA synthesis and Cas9-mediated region-of-interest (ROI) liberation. To facilitate quantitative comparisons between multiple samples, we additionally develop an R package to standardize differential analysis of any type of NOMe-seq data. We extensively benchmark guidedNOMe-seq in a proof-of-concept study, dissecting the interplay of ChAHP and CTCF on chromatin. In summary we present a cost-effective, scalable, and customizable target enrichment extension to the existing NOMe-seq protocol allowing genome-scale quantification of nucleosome occupancy and transcription factor binding at single allele resolution.
Asunto(s)
Alelos , Cromatina , Secuenciación de Nucleótidos de Alto Rendimiento , Nucleosomas , Cromatina/genética , Cromatina/metabolismo , Nucleosomas/genética , Nucleosomas/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Análisis de Secuencia de ADN/métodosRESUMEN
In eukaryotes, repetitive DNA can become silenced de novo, either transcriptionally or post-transcriptionally, by processes independent of strong sequence-specific cues. The mechanistic nature of such processes remains poorly understood. We found that in the fungus Neurospora crassa, de novo initiation of both transcriptional and post-transcriptional silencing was linked to perturbed chromatin, which was produced experimentally by the aberrant activity of transcription factors at the tetO operator array. Transcriptional silencing was mediated by canonical constitutive heterochromatin. On the other hand, post-transcriptional silencing resembled repeat-induced quelling but occurred normally when homologous recombination was inactivated. All silencing of the tetO array was dependent on SAD-6, fungal ortholog of the SWI/SNF chromatin remodeler ATRX (Alpha Thalassemia/Mental Retardation Syndrome X-Linked), which was required to maintain nucleosome occupancy at the perturbed locus. In addition, we found that two other types of sequences (the lacO array and native AT-rich DNA) could also undergo recombination-independent quelling associated with perturbed chromatin. These results suggested a model in which the de novo initiation of transcriptional and post-transcriptional silencing is coupled to the remodeling of perturbed chromatin.
Asunto(s)
Ensamble y Desensamble de Cromatina , Silenciador del Gen , Neurospora crassa , Transcripción Genética , Neurospora crassa/genética , Neurospora crassa/metabolismo , Cromatina/metabolismo , Cromatina/genética , Heterocromatina/metabolismo , Heterocromatina/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Nucleosomas/metabolismo , Nucleosomas/genéticaRESUMEN
Eukaryotic genomes are organized into 3D structures, which range from small-scale nucleosome arrays to large-scale chromatin domains. These structures have an important role in the regulation of transcription and other nuclear processes. Despite advances in our understanding of the properties, functions, and underlying mechanisms of genome structures, there are many open questions about the interplay between these structures across scales. In particular, it is not well understood if and how 1D features of nucleosome arrays influence large-scale 3D genome folding patterns. In this review, we discuss recent studies that address these questions and summarize our current understanding of the relationship between nucleosome positioning and higher-order genome folding.
Asunto(s)
Genoma , Nucleosomas , Nucleosomas/metabolismo , Nucleosomas/química , Humanos , Animales , Ensamble y Desensamble de Cromatina , Conformación de Ácido Nucleico , Cromatina/metabolismo , Cromatina/químicaRESUMEN
Yeast COMPASS (complex of proteins associated with Set1) and human MLL (mixed-lineage leukemia) complexes are histone H3 lysine 4 methyltransferases with critical roles in gene regulation and embryonic development. Both complexes share a conserved C-terminal SET domain, responsible for catalyzing histone H3 K4 methylation on nucleosomes. Notably, their catalytic activity toward nucleosomes is enhanced and optimized with assembly of auxiliary subunits. In this review, we aim to illustrate the recent X-ray and cryo-EM structures of yeast COMPASS and human MLL1 core complexes bound to either unmodified nucleosome core particle (NCP) or H2B mono-ubiquitinated NCP (H2Bub.NCP). We further delineate how each auxiliary component of the complex contributes to the NCP and ubiquitin recognition to maximize the methyltransferase activity.
Asunto(s)
N-Metiltransferasa de Histona-Lisina , Proteína de la Leucemia Mieloide-Linfoide , Nucleosomas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Humanos , Nucleosomas/metabolismo , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/química , Proteína de la Leucemia Mieloide-Linfoide/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Histonas/metabolismo , Histonas/química , Histonas/genética , Microscopía por Crioelectrón/métodosRESUMEN
50th Anniversary of the Nucleosome Discovery.
Asunto(s)
Nucleosomas , Nucleosomas/metabolismo , Historia del Siglo XX , Historia del Siglo XXI , Aniversarios y Eventos Especiales , HumanosRESUMEN
The chemical synthesis of homogeneously ubiquitylated histones is a powerful approach to decipher histone ubiquitylation-dependent epigenetic regulation. Among the various methods, α-halogen ketone-mediated conjugation chemistry has recently been an attractive strategy to generate single-monoubiquitylated histones for biochemical and structural studies. Herein, we report the use of this strategy to prepare not only dual- and even triple-monoubiquitylated histones but also diubiquitin-modified histones. We were surprised to find that the synthetic efficiencies of multi-monoubiquitylated histones were comparable to those of single-monoubiquitylated ones, suggesting that this strategy is highly tolerant to the number of ubiquitin monomers installed onto histones. The facile generation of a series of single-, dual-, and triple-monoubiquitylated H3 proteins enabled us to evaluate the influence of ubiquitylation patterns on the binding of DNA methyltransferase 1 (DNMT1) to nucleosomes. Our study highlights the potential of site-specific conjugation chemistry to generate chemically defined histones for epigenetic studies.
Asunto(s)
Histonas , Cetonas , Ubiquitinación , Histonas/química , Histonas/metabolismo , Histonas/síntesis química , Cetonas/química , Ubiquitina/química , Humanos , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/química , Nucleosomas/química , Nucleosomas/metabolismoRESUMEN
In addition to replicative histones, eukaryotic genomes encode a repertoire of non-replicative variant histones, providing additional layers of structural and epigenetic regulation. Here, we systematically replace individual replicative human histones with non-replicative human variant histones using a histone replacement system in yeast. We show that variants H2A.J, TsH2B, and H3.5 complement their respective replicative counterparts. However, macroH2A1 fails to complement, and its overexpression is toxic in yeast, negatively interacting with yeast's native histones and kinetochore genes. To isolate yeast with macroH2A1 chromatin, we uncouple the effects of its macro and histone fold domains, revealing that both domains suffice to override native nucleosome positioning. Furthermore, both uncoupled constructs of macroH2A1 exhibit lower nucleosome occupancy, decreased short-range chromatin interactions (<20 kb), disrupted centromeric clustering, and increased chromosome instability. Our observations demonstrate that lack of a canonical histone H2A dramatically alters chromatin organization in yeast, leading to genome instability and substantial fitness defects.
Asunto(s)
Inestabilidad Genómica , Histonas , Nucleosomas , Saccharomyces cerevisiae , Humanos , Centrómero/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Cinetocoros/metabolismo , Nucleosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
The histone H2A variant H2A.W occupies transposons and thus prevents access to them in Arabidopsis thaliana. H2A.W is deposited by the chromatin remodeler DDM1, which also promotes the accessibility of chromatin writers to heterochromatin by an unknown mechanism. To shed light on this question, we solve the cryo-EM structures of nucleosomes containing H2A and H2A.W, and the DDM1-H2A.W nucleosome complex. These structures show that the DNA end flexibility of the H2A nucleosome is higher than that of the H2A.W nucleosome. In the DDM1-H2A.W nucleosome complex, DDM1 binds to the N-terminal tail of H4 and the nucleosomal DNA and increases the DNA end flexibility of H2A.W nucleosomes. Based on these biochemical and structural results, we propose that DDM1 counters the low accessibility caused by nucleosomes containing H2A.W to enable the maintenance of repressive epigenetic marks on transposons and prevent their activity.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Ensamble y Desensamble de Cromatina , Microscopía por Crioelectrón , Histonas , Nucleosomas , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/química , Nucleosomas/metabolismo , Nucleosomas/ultraestructura , Nucleosomas/química , Histonas/metabolismo , Histonas/genética , Histonas/química , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/química , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Unión Proteica , Modelos Moleculares , ADN de Plantas/metabolismo , ADN de Plantas/genéticaRESUMEN
Micrococcal nuclease sequencing is the state-of-the-art method for determining chromatin structure and nucleosome positioning. Data analysis is complex due to the AT-dependent sequence bias of the endonuclease and the requirement for high sequencing depth. Here, we present the nucleosome-based MNase accessibility (nucMACC) pipeline unveiling the regulatory chromatin landscape by measuring nucleosome accessibility and stability. The nucMACC pipeline represents a systematic and genome-wide approach for detecting unstable ("fragile") nucleosomes. We have characterized the regulatory nucleosome landscape in Drosophila melanogaster, Saccharomyces cerevisiae, and mammals. Two functionally distinct sets of promoters were characterized, one associated with an unstable nucleosome and the other being nucleosome depleted. We show that unstable nucleosomes present intermediate states of nucleosome remodeling, preparing inducible genes for transcriptional activation in response to stimuli or stress. The presence of unstable nucleosomes correlates with RNA polymerase II proximal pausing. The nucMACC pipeline offers unparalleled precision and depth in nucleosome research and is a valuable tool for future nucleosome studies.