RESUMEN
Chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (-seq) has been the most common genomics method for studying DNA-protein interactions in the last decade. ChIP-seq technology became standard both experimentally and computationally. This chapter presents a core workflow that covers data processing and initial analytical steps of ChIP-seq data. We provide a step-by-step protocol of the commands as well as a fully assembled Snakemake workflow. Along the protocol, we discuss key tool parameters, quality control, output reports, and preliminary results.
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Secuenciación de Inmunoprecipitación de Cromatina , Biología Computacional , Programas Informáticos , Flujo de Trabajo , Secuenciación de Inmunoprecipitación de Cromatina/métodos , Biología Computacional/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Datos , Inmunoprecipitación de Cromatina/métodos , HumanosRESUMEN
Direct neuronal reprogramming is a promising approach to regenerate neurons from local glial cells. However, mechanisms of epigenome remodeling and co-factors facilitating this process are unclear. In this study, we combined single-cell multiomics with genome-wide profiling of three-dimensional nuclear architecture and DNA methylation in mouse astrocyte-to-neuron reprogramming mediated by Neurogenin2 (Ngn2) and its phosphorylation-resistant form (PmutNgn2), respectively. We show that Ngn2 drives multilayered chromatin remodeling at dynamic enhancer-gene interaction sites. PmutNgn2 leads to higher reprogramming efficiency and enhances epigenetic remodeling associated with neuronal maturation. However, the differences in binding sites or downstream gene activation cannot fully explain this effect. Instead, we identified Yy1, a transcriptional co-factor recruited by direct interaction with Ngn2 to its target sites. Upon deletion of Yy1, activation of neuronal enhancers, genes and ultimately reprogramming are impaired without affecting Ngn2 binding. Thus, our work highlights the key role of interactors of proneural factors in direct neuronal reprogramming.
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Astrocitos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Reprogramación Celular , Proteínas del Tejido Nervioso , Neuronas , Factor de Transcripción YY1 , Animales , Factor de Transcripción YY1/metabolismo , Factor de Transcripción YY1/genética , Astrocitos/metabolismo , Ratones , Reprogramación Celular/fisiología , Neuronas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Epigenoma , Ensamble y Desensamble de Cromatina , Epigénesis Genética , Células CultivadasRESUMEN
DNA replication enables genetic inheritance across the kingdoms of life. Replication occurs with a defined temporal order known as the replication timing (RT) programme, leading to organization of the genome into early- or late-replicating regions. RT is cell-type specific, is tightly linked to the three-dimensional nuclear organization of the genome1,2 and is considered an epigenetic fingerprint3. In spite of its importance in maintaining the epigenome4, the developmental regulation of RT in mammals in vivo has not been explored. Here, using single-cell Repli-seq5, we generated genome-wide RT maps of mouse embryos from the zygote to the blastocyst stage. Our data show that RT is initially not well defined but becomes defined progressively from the 4-cell stage, coinciding with strengthening of the A and B compartments. We show that transcription contributes to the precision of the RT programme and that the difference in RT between the A and B compartments depends on RNA polymerase II at zygotic genome activation. Our data indicate that the establishment of nuclear organization precedes the acquisition of defined RT features and primes the partitioning of the genome into early- and late-replicating domains. Our work sheds light on the establishment of the epigenome at the beginning of mammalian development and reveals the organizing principles of genome organization.
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Momento de Replicación del ADN , Embrión de Mamíferos , Genoma , Animales , Ratones , Blastocisto/citología , Blastocisto/metabolismo , Cromatina/genética , Epigenoma/genética , Genoma/genética , ARN Polimerasa II/metabolismo , Cigoto/citología , Cigoto/crecimiento & desarrollo , Cigoto/metabolismo , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismoRESUMEN
Fertilization in mammals is accompanied by an intense period of chromatin remodeling and major changes in nuclear organization. How the earliest events in embryogenesis, including zygotic genome activation (ZGA) during maternal-to-zygotic transition, influence such remodeling remains unknown. Here, we have investigated the establishment of nuclear architecture, focusing on the remodeling of lamina-associated domains (LADs) during this transition. We report that LADs reorganize gradually in two-cell embryos and that blocking ZGA leads to major changes in nuclear organization, including altered chromatin and genomic features of LADs and redistribution of H3K4me3 toward the nuclear lamina. Our data indicate that the rearrangement of LADs is an integral component of the maternal-to-zygotic transition and that transcription contributes to shaping nuclear organization at the beginning of mammalian development.
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ARN Polimerasa II , Transcripción Genética , Animales , Ratones , ARN Polimerasa II/genética , Desarrollo Embrionario/genética , Cigoto , Mamíferos/genética , Regulación del Desarrollo de la Expresión Génica , CromatinaRESUMEN
The majority of our genome is composed of repeated DNA sequences that assemble into heterochromatin, a highly compacted structure that constrains their mutational potential. How heterochromatin forms during development and how its structure is maintained are not fully understood. Here, we show that mouse heterochromatin phase-separates after fertilization, during the earliest stages of mammalian embryogenesis. Using high-resolution quantitative imaging and molecular biology approaches, we show that pericentromeric heterochromatin displays properties consistent with a liquid-like state at the two-cell stage, which change at the four-cell stage, when chromocenters mature and heterochromatin becomes silent. Disrupting the condensates results in altered transcript levels of pericentromeric heterochromatin, suggesting a functional role for phase separation in heterochromatin function. Thus, our work shows that mouse heterochromatin forms membrane-less compartments with biophysical properties that change during development and provides new insights into the self-organization of chromatin domains during mammalian embryogenesis.
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Cromatina , Heterocromatina , Animales , Ratones , Embrión de Mamíferos , Genoma , Mamíferos/genéticaRESUMEN
H4 lysine 20 dimethylation (H4K20me2) is the most abundant histone modification in vertebrate chromatin. It arises from sequential methylation of unmodified histone H4 proteins by the mono-methylating enzyme PR-SET7/KMT5A, followed by conversion to the dimethylated state by SUV4-20H (KMT5B/C) enzymes. We have blocked the deposition of this mark by depleting Xenopus embryos of SUV4-20H1/H2 methyltransferases. In the larval epidermis, this results in a severe loss of cilia in multiciliated cells (MCC), a key component of mucociliary epithelia. MCC precursor cells are correctly specified, amplify centrioles, but ultimately fail in ciliogenesis because of the perturbation of cytoplasmic processes. Genome-wide transcriptome profiling reveals that SUV4-20H1/H2-depleted ectodermal explants preferentially down-regulate the expression of several hundred ciliogenic genes. Further analysis demonstrated that knockdown of SUV4-20H1 alone is sufficient to generate the MCC phenotype and that its catalytic activity is needed for axoneme formation. Overexpression of the H4K20me1-specific histone demethylase PHF8/KDM7B also rescues the ciliogenic defect in a significant manner. Taken together, this indicates that the conversion of H4K20me1 to H4K20me2 by SUV4-20H1 is critical for the formation of cilia tufts.
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Cromatina , Histonas , Animales , Diferenciación Celular/genética , Histona Metiltransferasas/genética , Histona Metiltransferasas/metabolismo , Histonas/metabolismo , Xenopus laevis/genéticaRESUMEN
The chromatin environment at origins of replication is thought to influence DNA replication initiation in eukaryotic genomes. However, it remains unclear how and which chromatin features control the firing of early-efficient (EE) or late-inefficient (LI) origins. Here, we use site-specific recombination and single-locus chromatin isolation to purify EE and LI replication origins in Saccharomyces cerevisiae. Using mass spectrometry, we define the protein composition of native chromatin regions surrounding the EE and LI replication start sites. In addition to known origin interactors, we find the microtubule-binding Ask1/DASH complex as an origin-regulating factor. Strikingly, tethering of Ask1 to individual origin sites advances replication timing (RT) of the targeted chromosomal domain. Targeted degradation of Ask1 globally changes RT of a subset of origins, which can be reproduced by inhibiting microtubule dynamics. Thus, our findings mechanistically connect RT and chromosomal organization via Ask1/DASH with the microtubule cytoskeleton.
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Proteínas Asociadas a Microtúbulos , Origen de Réplica , Proteínas de Saccharomyces cerevisiae , Cromatina/metabolismo , ADN/metabolismo , Replicación del ADN , Momento de Replicación del ADN , Proteínas Asociadas a Microtúbulos/metabolismo , Complejos Multiproteicos/metabolismo , Proteómica , Origen de Réplica/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
How transcription is regulated as development commences is fundamental to understand how the transcriptionally silent mature gametes are reprogrammed. The embryonic genome is activated for the first time during zygotic genome activation (ZGA). How RNA polymerase II (Pol II) and productive elongation are regulated during this process remains elusive. Here, we generate genome-wide maps of Serine 5 and Serine 2-phosphorylated Pol II during and after ZGA in mouse embryos. We find that both phosphorylated Pol II forms display similar distributions across genes during ZGA, with typical elongation enrichment of Pol II emerging after ZGA. Serine 2-phosphorylated Pol II occurs at genes prior to their activation, suggesting that Serine 2 phosphorylation may prime gene expression. Functional perturbations demonstrate that CDK9 and SPT5 are major ZGA regulators and that SPT5 prevents precocious activation of some genes. Overall, our work sheds molecular insights into transcriptional regulation at the beginning of mammalian development.
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ARN Polimerasa II , Cigoto , Ratones , Animales , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Cigoto/metabolismo , Fosforilación , Genoma , Serina/metabolismo , Activación Transcripcional , Regulación del Desarrollo de la Expresión Génica , Mamíferos/metabolismoRESUMEN
The oligodendrocyte progenitors (OPCs) are at the front of the glial reaction to the traumatic brain injury. However, regulatory pathways steering the OPC reaction as well as the role of reactive OPCs remain largely unknown. Here, we compared a long-lasting, exacerbated reaction of OPCs to the adult zebrafish brain injury with a timely restricted OPC activation to identify the specific molecular mechanisms regulating OPC reactivity and their contribution to regeneration. We demonstrated that the influx of the cerebrospinal fluid into the brain parenchyma after injury simultaneously activates the toll-like receptor 2 (Tlr2) and the chemokine receptor 3 (Cxcr3) innate immunity pathways, leading to increased OPC proliferation and thereby exacerbated glial reactivity. These pathways were critical for long-lasting OPC accumulation even after the ablation of microglia and infiltrating monocytes. Importantly, interference with the Tlr1/2 and Cxcr3 pathways after injury alleviated reactive gliosis, increased new neuron recruitment, and improved tissue restoration.
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Células Precursoras de Oligodendrocitos , Animales , Encéfalo , Gliosis/metabolismo , Inmunidad Innata , Células Precursoras de Oligodendrocitos/metabolismo , Pez CebraRESUMEN
Numerous chromatin remodeling enzymes position nucleosomes in eukaryotic cells. Aside from these factors, transcription, DNA sequence, and statistical positioning of nucleosomes also shape the nucleosome landscape. The precise contributions of these processes remain unclear due to their functional redundancy in vivo. By incisive genome engineering, we radically decreased their redundancy in Saccharomyces cerevisiae. The transcriptional machinery strongly disrupts evenly spaced nucleosomes. Proper nucleosome density and DNA sequence are critical for their biogenesis. The INO80 remodeling complex helps space nucleosomes in vivo and positions the first nucleosome over genes in an H2A.Z-independent fashion. INO80 requires its Arp8 subunit but unexpectedly not the Nhp10 module for spacing. Cells with irregularly spaced nucleosomes suffer from genotoxic stress including DNA damage, recombination and transpositions. We derive a model of the biogenesis of the nucleosome landscape and suggest that it evolved not only to regulate but also to protect the genome.
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Cromatina , Epigenómica , Nucleosomas/fisiología , Ensamble y Desensamble de Cromatina , ADN , Daño del ADN , Ingeniería , Células Eucariotas , Proteínas del Grupo de Alta Movilidad/metabolismo , Histonas , Proteínas de Microfilamentos/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae , Factores de TranscripciónRESUMEN
Heterochromatin formation requires three distinct steps: nucleation, self-propagation (spreading) along the chromosome, and faithful maintenance after each replication cycle. Impeding any of those steps induces heterochromatin defects and improper gene expression. The essential histone chaperone FACT (facilitates chromatin transcription) has been implicated in heterochromatin silencing, but the mechanisms by which FACT engages in this process remain opaque. Here, we pinpoint its function to the heterochromatin spreading process in fission yeast. FACT impairment reduces nucleation-distal H3K9me3 and HP1/Swi6 accumulation at subtelomeres and derepresses genes in the vicinity of heterochromatin boundaries. FACT promotes spreading by repressing heterochromatic histone turnover, which is crucial for the H3K9me2 to me3 transition that enables spreading. FACT mutant spreading defects are suppressed by removal of the H3K9 methylation antagonist Epe1. Together, our study identifies FACT as a histone chaperone that promotes heterochromatin spreading and lends support to the model that regulated histone turnover controls the propagation of repressive methylation marks.
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Aminopeptidasas/metabolismo , Ensamble y Desensamble de Cromatina , Heterocromatina/metabolismo , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Aminopeptidasas/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Regulación Fúngica de la Expresión Génica , Silenciador del Gen , Heterocromatina/genética , Chaperonas de Histonas/genética , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Metilación , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Transcripción GenéticaRESUMEN
Postzygotic isolation by genomic conflict is a major cause for the formation of species. Despite its importance, the molecular mechanisms that result in the lethality of interspecies hybrids are still largely unclear. The genus Drosophila, which contains over 1600 different species, is one of the best characterized model systems to study these questions. We showed in the past that the expression levels of the two hybrid incompatibility factors Hmr and Lhr diverged in the two closely related Drosophila species, D. melanogaster and D. simulans, resulting in an increased level of both proteins in interspecies hybrids. The overexpression of the two proteins also leads to mitotic defects, a misregulation in the expression of transposable elements and decreased fertility in pure species. In this work, we describe a distinct six subunit protein complex containing HMR and LHR and analyse the effect of Hmr mutations on complex integrity and function. Our experiments suggest that HMR needs to bring together components of centromeric and pericentromeric chromatin to fulfil its physiological function and to cause hybrid male lethality.
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Proteínas de Drosophila/genética , Aislamiento Reproductivo , Animales , Centrómero/metabolismo , Elementos Transponibles de ADN/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Drosophila simulans/genética , Drosophila simulans/metabolismo , Genes Letales/genética , Especiación Genética , Hibridación Genética/genética , Reproducción/genéticaRESUMEN
RNA-protein interactions are often mediated by dedicated canonical RNA binding domains. However, interactions through non-canonical domains with unknown specificity are increasingly observed, raising the question how RNA targets are recognized. Knowledge of the intrinsic RNA binding specificity contributes to the understanding of target selectivity and function of an individual protein. The presented in vitro RNA immunoprecipitation assay (vitRIP) uncovers intrinsic RNA binding specificities of isolated proteins using the total cellular RNA pool as a library. Total RNA extracted from cells or tissues is incubated with purified recombinant proteins, RNA-protein complexes are immunoprecipitated and bound transcripts are identified by deep sequencing or quantitative RT-PCR. Enriched RNA classes and the nucleotide frequency in these RNAs inform on the intrinsic specificity of the recombinant protein. The simple and versatile protocol can be adapted to other RNA binding proteins and total RNA libraries from any cell type or tissue. Graphic abstract: Figure 1. Schematic of the in vitro RNA immunoprecipitation (vitRIP) protocol.
RESUMEN
Poly(ADP-ribose) polymerase (PARP) inhibitors are used in the treatment of BRCA-deficient cancers, with treatments currently extending toward other homologous recombination defective tumors. In a genome-wide CRISPR knockout screen with olaparib, we identify ALC1 (Amplified in Liver Cancer 1)-a cancer-relevant poly(ADP-ribose)-regulated chromatin remodeling enzyme-as a key modulator of sensitivity to PARP inhibitor. We found that ALC1 can remove inactive PARP1 indirectly through binding to PARylated chromatin. Consequently, ALC1 deficiency enhances trapping of inhibited PARP1, which then impairs the binding of both nonhomologous end-joining and homologous recombination repair factors to DNA lesions. We also establish that ALC1 overexpression, a common feature in multiple tumor types, reduces the sensitivity of BRCA-deficient cells to PARP inhibitors. Together, we conclude that ALC1-dependent PARP1 mobilization is a key step underlying PARP inhibitor resistance.
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Cromatina , ADN Helicasas , Proteínas de Unión al ADN , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Línea Celular Tumoral , Cromatina/genética , Ensamble y Desensamble de Cromatina , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismoRESUMEN
The MLE DExH helicase and the roX lncRNAs are essential components of the chromatin modifying Dosage Compensation Complex (DCC) in Drosophila. To explore the mechanism of ribonucleoprotein complex assembly, we developed vitRIP, an unbiased, transcriptome-wide in vitro assay that reveals RNA binding specificity. We found that MLE has intrinsic specificity for U-/A-rich sequences and tandem stem-loop structures and binds many RNAs beyond roX in vitro. The selectivity of the helicase for physiological substrates is further enhanced by the core DCC. Unwinding of roX2 by MLE induces a highly selective RNA binding surface in the unstructured C-terminus of the MSL2 subunit and triggers-specific association of MLE and roX2 with the core DCC. The exquisite selectivity of roX2 incorporation into the DCC thus originates from intimate cooperation between the helicase and the core DCC involving two distinct RNA selection principles and their mutual refinement.
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Ensamble y Desensamble de Cromatina , ARN Largo no Codificante/metabolismo , Transcriptoma , Animales , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Clonación Molecular/métodos , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Unión Proteica , ARN Largo no Codificante/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Histone acetylation and deposition of H2A.Z variant are integral aspects of active transcription. In Drosophila, the single DOMINO chromatin regulator complex is thought to combine both activities via an unknown mechanism. Here we show that alternative isoforms of the DOMINO nucleosome remodeling ATPase, DOM-A and DOM-B, directly specify two distinct multi-subunit complexes. Both complexes are necessary for transcriptional regulation but through different mechanisms. The DOM-B complex incorporates H2A.V (the fly ortholog of H2A.Z) genome-wide in an ATP-dependent manner, like the yeast SWR1 complex. The DOM-A complex, instead, functions as an ATP-independent histone acetyltransferase complex similar to the yeast NuA4, targeting lysine 12 of histone H4. Our work provides an instructive example of how different evolutionary strategies lead to similar functional separation. In yeast and humans, nucleosome remodeling and histone acetyltransferase complexes originate from gene duplication and paralog specification. Drosophila generates the same diversity by alternative splicing of a single gene.
Cells contain a large number of proteins that control the activity of genes in response to various signals and changes in their environment. Often these proteins work together in groups called complexes. In the fruit fly Drosophila melanogaster, one of these complexes is called DOMINO. The DOMINO complex alters gene activity by interacting with other proteins called histones which influence how the genes are packaged and accessed within the cell. DOMINO works in two separate ways. First, it can replace certain histones with other variants that regulate genes differently. Second, it can modify histones by adding a chemical marker to them, which alters how they interact with genes. It was not clear how DOMINO can do both of these things and how that is controlled; but it was known that cells can make two different forms of the central component of the complex, called DOM-A and DOM-B, which are both encoded by the same gene. Scacchetti et al. have now studied fruit flies to understand the activities of these forms. This revealed that they do have different roles and that gene activity in cells changes if either one is lost. The two forms operate as part complexes with different compositions and only DOM-A includes the TIP60 enzyme that is needed to modify histones. As such, it seems that DOM-B primarily replaces histones with variant forms, while DOM-A modifies existing histones. This means that each form has a unique role associated with each of the two known behaviors of this complex. The presence of two different DOMINO complexes is common to flies and, probably, other insects. Yet, in other living things, such as mammals and yeast, their two roles are carried out by protein complexes originating from two distinct genes. This illustrates a concept called convergent evolution, where different organisms find different solutions for the same problem. As such, these findings provide an insight into the challenges encountered through evolution and the diverse solutions that have developed. They will also help us to understand the ways in which protein activities can adapt to different needs over evolutionary time.
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Proteínas de Drosophila/metabolismo , Drosophila/enzimología , Histona Acetiltransferasas/metabolismo , Complejos Multiproteicos/metabolismo , Factores de Transcripción/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Ensamble y Desensamble de Cromatina , Drosophila/genética , Proteínas de Drosophila/genética , Histona Acetiltransferasas/genética , Histonas/genética , Histonas/metabolismo , Complejos Multiproteicos/genética , Nucleosomas/genética , Nucleosomas/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genéticaRESUMEN
During interphase centromeres often coalesce into a small number of chromocenters, which can be visualized as distinct, DAPI dense nuclear domains. Intact chromocenters play a major role in maintaining genome stability as they stabilize the transcriptionally silent state of repetitive DNA while ensuring centromere function. Despite its biological importance, relatively little is known about the molecular composition of the chromocenter or the processes that mediate chromocenter formation and maintenance. To provide a deeper molecular insight into the composition of the chromocenter and to demonstrate the usefulness of proximity-based biotinylation as a tool to investigate those questions, we performed super resolution microscopy and proximity-based biotinylation experiments of three distinct proteins associated with the chromocenter in Drosophila. Our work revealed an intricate internal architecture of the chromocenter suggesting a complex multilayered structure of this intranuclear domain.
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Proteína A Centromérica/metabolismo , Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Drosophila/metabolismo , Interfase/genética , Adenosina Trifosfatasas/metabolismo , Animales , Biotinilación , Proteínas de Ciclo Celular/análisis , Línea Celular , Núcleo Celular/metabolismo , Proteína A Centromérica/genética , Cromatina/metabolismo , Secuenciación de Inmunoprecipitación de Cromatina , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/análisis , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Complejos Multiproteicos/metabolismo , Proteómica , Proteínas Recombinantes de Fusión/análisis , CohesinasRESUMEN
The histone chaperone FACT and histone H2B ubiquitination (H2Bub) facilitate RNA polymerase II (Pol II) passage through chromatin, yet it is not clear how they cooperate mechanistically. We used genomics, genetic, biochemical, and microscopic approaches to dissect their interplay in Schizosaccharomyces pombe. We show that FACT and H2Bub globally repress antisense transcripts near the 5' end of genes and inside gene bodies, respectively. The accumulation of these transcripts is accompanied by changes at genic nucleosomes and Pol II redistribution. H2Bub is required for FACT activity in genic regions. In the H2Bub mutant, FACT binding to chromatin is altered and its association with histones is stabilized, which leads to the reduction of genic nucleosomes. Interestingly, FACT depletion globally restores nucleosomes in the H2Bub mutant. Moreover, in the absence of Pob3, the FACT Spt16 subunit controls the 3' end of genes. Furthermore, FACT maintains nucleosomes in subtelomeric regions, which is crucial for their compaction.
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Proteínas de Unión al ADN/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Factores de Elongación Transcripcional/metabolismo , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas del Grupo de Alta Movilidad/genética , Histonas/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Nucleosomas/metabolismo , Unión Proteica , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Factores de Transcripción/metabolismo , Factores de Elongación Transcripcional/genética , UbiquitinaciónRESUMEN
In flies, the chromosomal kinase JIL-1 is responsible for most interphase histone H3S10 phosphorylation and has been proposed to protect active chromatin from acquiring heterochromatic marks, such as dimethylated histone H3K9 (H3K9me2) and HP1. Here, we show that JIL-1's targeting to chromatin depends on a PWWP domain-containing protein JASPer (JIL-1 Anchoring and Stabilizing Protein). JASPer-JIL-1 (JJ)-complex is the major form of kinase in vivo and is targeted to active genes and telomeric transposons via binding of the PWWP domain of JASPer to H3K36me3 nucleosomes, to modulate transcriptional output. JIL-1 and JJ-complex depletion in cycling cells lead to small changes in H3K9me2 distribution at active genes and telomeric transposons. Finally, we identify interactors of the endogenous JJ-complex and propose that JIL-1 not only prevents heterochromatin formation but also coordinates chromatin-based regulation in the transcribed part of the genome.
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Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Histonas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , Interfase , Metilación , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
In Drosophila melanogaster males, X-chromosome monosomy is compensated by chromosome-wide transcription activation. We found that complete dosage compensation during embryogenesis takes surprisingly long and is incomplete even after 10 h of development. Although the activating dosage compensation complex (DCC) associates with the X-chromosome and MOF acetylates histone H4 early, many genes are not compensated. Acetylation levels on gene bodies continue to increase for several hours after gastrulation in parallel with progressive compensation. Constitutive genes are compensated earlier than developmental genes. Remarkably, later compensation correlates with longer distances to DCC binding sites. This time-space relationship suggests that DCC action on target genes requires maturation of the active chromosome compartment.