RESUMEN
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.
Asunto(s)
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.
Asunto(s)
Cromatina , Heterocromatina , Animales , Ratones , Embrión de Mamíferos , Genoma , Mamíferos/genéticaRESUMEN
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.
Asunto(s)
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
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.
Asunto(s)
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 mammals, the emergence of totipotency after fertilization involves extensive rearrangements of the spatial positioning of the genome1,2. However, the contribution of spatial genome organization to the regulation of developmental programs is unclear3. Here we generate high-resolution maps of genomic interactions with the nuclear lamina (a filamentous meshwork that lines the inner nuclear membrane) in mouse pre-implantation embryos. We reveal that nuclear organization is not inherited from the maternal germline but is instead established de novo shortly after fertilization. The two parental genomes establish lamina-associated domains (LADs)4 with different features that converge after the 8-cell stage. We find that the mechanism of LAD establishment is unrelated to DNA replication. Instead, we show that paternal LAD formation in zygotes is prevented by ectopic expression of Kdm5b, which suggests that LAD establishment may be dependent on remodelling of H3K4 methylation. Our data suggest a step-wise assembly model whereby early LAD formation precedes consolidation of topologically associating domains.
Asunto(s)
Posicionamiento de Cromosoma , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Genoma/fisiología , Lámina Nuclear/metabolismo , Animales , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/embriología , Desarrollo Embrionario , Femenino , Fertilización , Histona Demetilasas con Dominio de Jumonji/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Oocitos/citología , Oocitos/metabolismo , Cigoto/citología , Cigoto/metabolismoRESUMEN
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.
Asunto(s)
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
Multicellular organisms depend on cell-type-specific division of labor for survival. Specific cell types have their unique developmental program and respond differently to environmental challenges, yet are orchestrated by the same genetic blueprint. A key challenge in biology is thus to understand how genes are expressed in the right place, at the right time, and to the right level. Further, this exquisite control of gene expression is perturbed in many diseases. As a consequence, coordinated physiological responses to the environment are compromised. Recently, innovative tools have been developed that are able to capture genome-wide gene expression using cell-type-specific approaches. These novel techniques allow us to understand gene regulation in vivo with unprecedented resolution and give us mechanistic insights into how multicellular organisms adapt to changing environments. In this article, we discuss the considerations needed when designing your own cell-type-specific experiment from the isolation of your starting material through selecting the appropriate controls and validating the data.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Genoma/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Especificidad de Órganos/genética , Análisis de la Célula Individual/métodos , Animales , Humanos , Reproducibilidad de los ResultadosRESUMEN
The rules defining which small fraction of related DNA sequences can be selectively bound by a transcription factor are poorly understood. One of the most challenging tasks in DNA recognition is posed by dosage compensation systems that require the distinction between sex chromosomes and autosomes. In Drosophila melanogaster, the male-specific lethal dosage compensation complex (MSL-DCC) doubles the level of transcription from the single male X chromosome, but the nature of this selectivity is not known. Previous efforts to identify X-chromosome-specific target sequences were unsuccessful as the identified MSL recognition elements lacked discriminative power. Therefore, additional determinants such as co-factors, chromatin features, RNA and chromosome conformation have been proposed to refine targeting further. Here, using an in vitro genome-wide DNA binding assay, we show that recognition of the X chromosome is an intrinsic feature of the MSL-DCC. MSL2, the male-specific organizer of the complex, uses two distinct DNA interaction surfaces-the CXC and proline/basic-residue-rich domains-to identify complex DNA elements on the X chromosome. Specificity is provided by the CXC domain, which binds a novel motif defined by DNA sequence and shape. This motif characterizes a subclass of MSL2-binding sites, which we name PionX (pioneering sites on the X) as they appeared early during the recent evolution of an X chromosome in D. miranda and are the first chromosomal sites to be bound during de novo MSL-DCC assembly. Our data provide the first, to our knowledge, documented molecular mechanism through which the dosage compensation machinery distinguishes the X chromosome from an autosome. They highlight fundamental principles in the recognition of complex DNA elements by protein that will have a strong impact on many aspects of chromosome biology.
Asunto(s)
Compensación de Dosificación (Genética)/genética , Drosophila melanogaster/genética , Complejos Multiproteicos/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Cromosoma X/genética , Secuencias de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Evolución Molecular , Femenino , Genoma de los Insectos/genética , Masculino , Complejos Multiproteicos/química , Proteínas Nucleares/metabolismo , Conformación de Ácido Nucleico , Motivos de Nucleótidos , Dominios Proteicos , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Especificidad por Sustrato , Factores de Transcripción/metabolismo , Cromosoma X/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.
Asunto(s)
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
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.
Asunto(s)
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
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.
Asunto(s)
Cromosomas de Insectos , Compensación de Dosificación (Genética) , Drosophila melanogaster/genética , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Cromosoma X , Acetilación , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Embrión no Mamífero , Femenino , Gastrulación/genética , Dosificación de Gen , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Histonas/genética , Histonas/metabolismo , Masculino , Monosomía , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Activación TranscripcionalRESUMEN
Preblastoderm Drosophila embryo development is characterized by fast cycles of nuclear divisions. Extracts from these embryos can be used to reconstitute complex chromatin with high efficiency. We now discovered that this chromatin assembly system contains activities that recognize unprotected DNA ends and signal DNA damage through phosphorylation. DNA ends are initially bound by Ku and MRN complexes. Within minutes, the phosphorylation of H2A.V (homologous to γH2A.X) initiates from DNA breaks and spreads over tens of thousands DNA base pairs. The γH2A.V phosphorylation remains tightly associated with the damaged DNA and does not spread to undamaged DNA in the same reaction. This first observation of long-range γH2A.X spreading along damaged chromatin in an in vitro system provides a unique opportunity for mechanistic dissection. Upon further incubation, DNA ends are rendered single-stranded and bound by the RPA complex. Phosphoproteome analyses reveal damage-dependent phosphorylation of numerous DNA-end-associated proteins including Ku70, RPA2, CHRAC16, the exonuclease Rrp1 and the telomer capping complex. Phosphorylation of spindle assembly checkpoint components and of microtubule-associated proteins required for centrosome integrity suggests this cell-free system recapitulates processes involved in the regulated elimination of fatally damaged syncytial nuclei.
Asunto(s)
Sistema Libre de Células/metabolismo , Roturas del ADN , Drosophila/genética , Transducción de Señal , Animales , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Reparación del ADN , Drosophila/citología , Drosophila/embriología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Histonas/genética , Histonas/metabolismo , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Fosforilación , Proteoma/genética , Proteoma/metabolismo , Proteómica/métodosRESUMEN
Understanding the molecular basis of hybrid incompatibilities is a fundamental pursuit in evolutionary genetics. In crosses between Drosophila melanogaster females and Drosophila simulans males, an interaction between at least three genes is necessary for hybrid male lethality: Hmrâmel, Lhrâsim, and gfzfâsim. Although HMR and LHR physically bind each other and function together in a single complex, the connection between gfzf and either of these proteins remains mysterious. Here, we show that GFZF localizes to many regions of the genome in both D. melanogaster and D. simulans, including at telomeric retrotransposon repeats. We find that GFZF localization at telomeres is significantly different between these two species, reflecting the rapid evolution of telomeric retrotransposon copy number composition between the two species. Next, we show that GFZF and HMR normally do not colocalize in D. melanogaster. In interspecies hybrids, however, HMR shows extensive mis-localization to GFZF sites, thus uncovering a new molecular interaction between these hybrid incompatibility factors. We find that spreading of HMR to GFZF sites requires gfzfâsim but not Lhrâsim, suggesting distinct roles for these factors in the hybrid incompatibility. Finally, we find that overexpression of HMR and LHR within species is sufficient to mis-localize HMR to GFZF binding sites, indicating that HMR has a natural low affinity for GFZF sites. Together, these studies provide the first insights into the different properties of gfzf between D. melanogaster and D. simulans, and uncover a molecular interaction between gfzf and Hmr in the form of altered protein localization.
Asunto(s)
Proteínas Portadoras/metabolismo , Drosophila/metabolismo , Hibridación Genética , Aislamiento Reproductivo , Animales , Proteínas Portadoras/genética , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , MasculinoRESUMEN
X chromosome dosage compensation in Drosophila requires chromosome-wide coordination of gene activation. The male-specific lethal dosage compensation complex (DCC) identifies and binds to X-chromosomal high-affinity sites (HAS) from which it boosts transcription. A sub-class of HAS, PionX sites, represent first contacts on the X. Here, we explored the chromosomal interactions of representative PionX sites by high-resolution 4C and determined the global chromosome conformation by Hi-C in sex-sorted embryos. Male and female X chromosomes display similar nuclear architecture, concordant with clustered, constitutively active genes. PionX sites, like HAS, are evenly distributed in the active compartment and engage in short- and long-range interactions beyond compartment boundaries. Long-range, inter-domain interactions between DCC binding sites are stronger in males, suggesting that the complex refines chromatin organization. By de novo induction of DCC in female cells, we monitored the extent of activation surrounding PionX sites. This revealed a remarkable range of DCC action not only in linear proximity, but also at megabase distance if close in space, suggesting that DCC profits from pre-existing chromosome folding to activate genes.
RESUMEN
Old age is associated with a progressive decline of mitochondrial function and changes in nuclear chromatin. However, little is known about how metabolic activity and epigenetic modifications change as organisms reach their midlife. Here, we assessed how cellular metabolism and protein acetylation change during early aging in Drosophila melanogaster. Contrary to common assumptions, we find that flies increase oxygen consumption and become less sensitive to histone deacetylase inhibitors as they reach midlife. Further, midlife flies show changes in the metabolome, elevated acetyl-CoA levels, alterations in protein-notably histone-acetylation, as well as associated transcriptome changes. Based on these observations, we decreased the activity of the acetyl-CoA-synthesizing enzyme ATP citrate lyase (ATPCL) or the levels of the histone H4 K12-specific acetyltransferase Chameau. We find that these targeted interventions both alleviate the observed aging-associated changes and promote longevity. Our findings reveal a pathway that couples changes of intermediate metabolism during aging with the chromatin-mediated regulation of transcription and changes in the activity of associated enzymes that modulate organismal life span.
Asunto(s)
Drosophila melanogaster/metabolismo , Histonas/metabolismo , Longevidad , Procesamiento Proteico-Postraduccional , ATP Citrato (pro-S)-Liasa/genética , ATP Citrato (pro-S)-Liasa/metabolismo , Acetilación , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Histonas/genéticaRESUMEN
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.
Asunto(s)
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.
Asunto(s)
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
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.
Asunto(s)
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.
Asunto(s)
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.