RESUMEN
Pericentric heterochromatin is a critical component of chromosomes marked by histone H3 K9 (H3K9) methylation1-3. However, what recruits H3K9-specific histone methyltransferases to pericentric regions in vertebrates remains unclear4, as does why pericentric regions in different species share the same H3K9 methylation mark despite lacking highly conserved DNA sequences2,5. Here we show that zinc-finger proteins ZNF512 and ZNF512B specifically localize at pericentric regions through direct DNA binding. Notably, both ZNF512 and ZNF512B are sufficient to initiate de novo heterochromatin formation at ectopically targeted repetitive regions and pericentric regions, as they directly recruit SUV39H1 and SUV39H2 (SUV39H) to catalyse H3K9 methylation. SUV39H2 makes a greater contribution to H3K9 trimethylation, whereas SUV39H1 seems to contribute more to silencing, probably owing to its preferential association with HP1 proteins. ZNF512 and ZNF512B from different species can specifically target pericentric regions of other vertebrates, because the atypical long linker residues between the zinc-fingers of ZNF512 and ZNF512B offer flexibility in recognition of non-consecutively organized three-nucleotide triplets targeted by each zinc-finger. This study addresses two long-standing questions: how constitutive heterochromatin is initiated and how seemingly variable pericentric sequences are targeted by the same set of conserved machinery in vertebrates.
Asunto(s)
Centrómero , Evolución Molecular , Heterocromatina , N-Metiltransferasa de Histona-Lisina , Histonas , Motivos de Nucleótidos , Animales , Humanos , Ratones , Centrómero/genética , Centrómero/metabolismo , Pollos , Homólogo de la Proteína Chromobox 5 , Silenciador del Gen , Heterocromatina/metabolismo , Heterocromatina/química , Heterocromatina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/química , Histonas/metabolismo , Histonas/química , Anfioxos , Metilación , Petromyzon , Proteínas Represoras/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Serpientes , Xenopus laevis , Pez Cebra , Dedos de ZincRESUMEN
The SWI/SNF2 chromatin remodeling factor decreased DNA methylation 1 (DDM1) is essential for the silencing of transposable elements (TEs) in both euchromatic and heterochromatic regions. Here, we determined the cryo-EM structures of DDM1-nucleosomeH2A and DDM1-nucleosomeH2A.W complexes at near-atomic resolution in the presence of the ATP analog ADP-BeFx. The structures show that nucleosomal DNA is unwrapped more on the surface of the histone octamer containing histone H2A than that containing histone H2A.W. DDM1 embraces one DNA gyre of the nucleosome and interacts with the N-terminal tails of histone H4. Although we did not observe DDM1-H2A.W interactions in our structures, the results of the pull-down experiments suggest a direct interaction between DDM1 and the core region of histone H2A.W. Our work provides mechanistic insights into the heterochromatin remodeling process driven by DDM1 in plants.
Asunto(s)
Ensamble y Desensamble de Cromatina , Microscopía por Crioelectrón , Heterocromatina , Histonas , Nucleosomas , Unión Proteica , Nucleosomas/metabolismo , Nucleosomas/química , Heterocromatina/metabolismo , Heterocromatina/química , Histonas/metabolismo , Histonas/química , Histonas/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Modelos Moleculares , Arabidopsis/metabolismo , Arabidopsis/genética , ADN/metabolismo , ADN/química , Factores de Transcripción/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Sitios de UniónRESUMEN
Abnormal cell nuclear shapes are hallmarks of diseases, including progeria, muscular dystrophy, and many cancers. Experiments have shown that disruption of heterochromatin and increases in euchromatin lead to nuclear deformations, such as blebs and ruptures. However, the physical mechanisms through which chromatin governs nuclear shape are poorly understood. To investigate how heterochromatin and euchromatin might govern nuclear morphology, we studied chromatin microphase separation in a composite coarse-grained polymer and elastic shell simulation model. By varying chromatin density, heterochromatin composition, and heterochromatin-lamina interactions, we show how the chromatin phase organization may perturb nuclear shape. Increasing chromatin density stabilizes the lamina against large fluctuations. However, increasing heterochromatin levels or heterochromatin-lamina interactions enhances nuclear shape fluctuations by a "wetting"-like interaction. In contrast, fluctuations are insensitive to heterochromatin's internal structure. Our simulations suggest that peripheral heterochromatin accumulation could perturb nuclear morphology, while nuclear shape stabilization likely occurs through mechanisms other than chromatin microphase organization.
Asunto(s)
Núcleo Celular , Cromatina , Heterocromatina , Núcleo Celular/metabolismo , Heterocromatina/metabolismo , Heterocromatina/química , Cromatina/metabolismo , Cromatina/química , Polímeros/química , Polímeros/metabolismo , Eucromatina/metabolismo , Eucromatina/química , Humanos , Separación de FasesRESUMEN
Chromatin three-dimensional (3D) organization inside the cell nucleus determines the separation of euchromatin and heterochromatin domains. Their segregation results in the definition of active and inactive chromatin compartments, whereby the local concentration of associated proteins, RNA and DNA results in the formation of distinct subnuclear structures. Thus, chromatin domains spatially confined in a specific 3D nuclear compartment are expected to share similar epigenetic features and biochemical properties, in terms of accessibility and solubility. Based on this rationale, we developed the 4f-SAMMY-seq to map euchromatin and heterochromatin based on their accessibility and solubility, starting from as little as 10 000 cells. Adopting a tailored bioinformatic data analysis approach we reconstruct also their 3D segregation in active and inactive chromatin compartments and sub-compartments, thus recapitulating the characteristic properties of distinct chromatin states. A key novelty of the new method is the capability to map both the linear segmentation of open and closed chromatin domains, as well as their compartmentalization in one single experiment.
Asunto(s)
Eucromatina , Heterocromatina , Heterocromatina/química , Heterocromatina/metabolismo , Eucromatina/química , Eucromatina/metabolismo , Eucromatina/genética , Humanos , Cromatina/química , Cromatina/metabolismo , Cromatina/genética , Núcleo Celular/genética , Núcleo Celular/química , Núcleo Celular/metabolismo , ADN/química , ADN/metabolismo , AnimalesRESUMEN
BACKGROUND: Emerging infectious disease-causing RNA viruses, such as the SARS-CoV-2 and Ebola viruses, are thought to rely on bats as natural reservoir hosts. Since these zoonotic viruses pose a great threat to humans, it is important to characterize the bat genome from multiple perspectives. Unsupervised machine learning methods for extracting novel information from big sequence data without prior knowledge or particular models are highly desirable for obtaining unexpected insights. We previously established a batch-learning self-organizing map (BLSOM) of the oligonucleotide composition that reveals novel genome characteristics from big sequence data. RESULTS: In this study, using the oligonucleotide BLSOM, we conducted a comparative genomic study of humans and six bat species. BLSOM is an explainable-type machine learning algorithm that reveals the diagnostic oligonucleotides contributing to sequence clustering (self-organization). When unsupervised machine learning reveals unexpected and/or characteristic features, these features can be studied in more detail via the much simpler and more direct standard distribution map method. Based on this combined strategy, we identified the Mb-level enrichment of CG dinucleotide (Mb-level CpG islands) around the termini of bat long-scaffold sequences. In addition, a class of CG-containing oligonucleotides were enriched in the centromeric and pericentromeric regions of human chromosomes. Oligonucleotides longer than tetranucleotides often represent binding motifs for a wide variety of proteins (e.g., transcription factor binding sequences (TFBSs)). By analyzing the penta- and hexanucleotide composition, we observed the evident enrichment of a wide range of hexanucleotide TFBSs in centromeric and pericentromeric heterochromatin regions on all human chromosomes. CONCLUSION: Function of transcription factors (TFs) beyond their known regulation of gene expression (e.g., TF-mediated looping interactions between two different genomic regions) has received wide attention. The Mb-level TFBS and CpG islands are thought to be involved in the large-scale nuclear organization, such as centromere and telomere clustering. TFBSs, which are enriched in centromeric and pericentromeric heterochromatin regions, are thought to play an important role in the formation of nuclear 3D structures. Our machine learning-based analysis will help us to understand the differential features of nuclear 3D structures in the human and bat genomes.
Asunto(s)
COVID-19 , Quirópteros/genética , Genoma Humano/genética , SARS-CoV-2/fisiología , Animales , COVID-19/transmisión , Quirópteros/virología , Islas de CpG , Genómica/métodos , Heterocromatina/química , Heterocromatina/genética , Humanos , Conformación Molecular , Oligonucleótidos/química , Aprendizaje Automático no SupervisadoRESUMEN
BACKGROUND: Structural variants (SVs) constitute a large proportion of the genomic variation that results in phenotypic variation in plants. However, they are still a largely unexplored feature in most plant genomes. Here, we present the whole-genome landscape of SVs between two model legume Medicago truncatula ecotypes-Jemalong A17 and R108- that have been extensively used in various legume biology studies. RESULTS: To catalogue SVs, we first resolved the previously published R108 genome assembly (R108 v1.0) to chromosome-scale using 124 × Hi-C data, resulting in a high-quality genome assembly. The inter-chromosomal reciprocal translocations between chromosomes 4 and 8 were confirmed by performing syntenic analysis between the two genomes. Combined with the Hi-C data, it appears that these translocation events had a significant effect on chromatin organization. Using both whole-genome and short-read alignments, we identified the genomic landscape of SVs between the two genomes, some of which may account for several phenotypic differences, including their differential responses to aluminum toxicity and iron deficiency, and the development of different anthocyanin leaf markings. We also found extensive SVs within the nodule-specific cysteine-rich gene family which encodes antimicrobial peptides essential for terminal bacteroid differentiation during nitrogen-fixing symbiosis. CONCLUSIONS: Our results provide a near-complete R108 genome assembly and the first genomic landscape of SVs obtained by comparing two M. truncatula ecotypes. This may provide valuable genomic resources for the functional and molecular research of legume biology in the future.
Asunto(s)
Cromatina/genética , Genoma de Planta , Medicago truncatula/genética , Cromosomas de las Plantas , Elementos Transponibles de ADN , Ecotipo , Eucromatina/química , Eucromatina/genética , Genes de Plantas , Heterocromatina/química , Heterocromatina/genética , Medicago truncatula/fisiología , Fijación del Nitrógeno/genética , Filogenia , Secuenciación Completa del GenomaRESUMEN
The formation of a diploid zygote is a highly complex cellular process that is entirely controlled by maternal gene products stored in the egg cytoplasm. This highly specialized transcriptional program is tightly controlled at the chromatin level in the female germline. As an extreme case in point, the massive and specific ovarian expression of the essential thioredoxin Deadhead (DHD) is critically regulated in Drosophila by the histone demethylase Lid and its partner, the histone deacetylase complex Sin3A/Rpd3, via yet unknown mechanisms. Here, we identified Snr1 and Mod(mdg4) as essential for dhd expression and investigated how these epigenomic effectors act with Lid and Sin3A to hyperactivate dhd. Using Cut&Run chromatin profiling with a dedicated data analysis procedure, we found that dhd is intriguingly embedded in an H3K27me3/H3K9me3-enriched mini-domain flanked by DNA regulatory elements, including a dhd promoter-proximal element essential for its expression. Surprisingly, Lid, Sin3a, Snr1 and Mod(mdg4) impact H3K27me3 and this regulatory element in distinct manners. However, we show that these effectors activate dhd independently of H3K27me3/H3K9me3, and that dhd remains silent in the absence of these marks. Together, our study demonstrates an atypical and critical role for chromatin regulators Lid, Sin3A, Snr1 and Mod(mdg4) to trigger tissue-specific hyperactivation within a unique heterochromatin mini-domain.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiología , Heterocromatina/genética , Histona Demetilasas/metabolismo , Proteínas de la Membrana/genética , Proteínas de Unión al ARN/metabolismo , Complejo Correpresor Histona Desacetilasa y Sin3/metabolismo , Tiorredoxinas/genética , Factores de Transcripción/metabolismo , Animales , Epigenómica , Femenino , Regulación de la Expresión Génica , Heterocromatina/química , Histonas/metabolismo , Masculino , Herencia Materna , Especificidad de Órganos , Ovario/química , Regiones Promotoras Genéticas , Elementos Reguladores de la TranscripciónRESUMEN
Epialleles are meiotically heritable variations in expression states that are independent from changes in DNA sequence. Although they are common in plant genomes, their molecular origins are unknown. Here we show, using mutant and experimental populations, that epialleles in Arabidopsis thaliana that result from ectopic hypermethylation are due to feedback regulation of pathways that primarily function to maintain DNA methylation at heterochromatin. Perturbations to maintenance of heterochromatin methylation leads to feedback regulation of DNA methylation in genes. Using single base resolution methylomes from epigenetic recombinant inbred lines (epiRIL), we show that epiallelic variation is abundant in euchromatin, yet, associates with QTL primarily in heterochromatin regions. Mapping three-dimensional chromatin contacts shows that genes that are hotspots for ectopic hypermethylation have increases in contact frequencies with regions possessing H3K9me2. Altogether, these data show that feedback regulation of pathways that have evolved to maintain heterochromatin silencing leads to the origins of spontaneous hypermethylated epialleles.
Asunto(s)
Alelos , Arabidopsis/genética , Epigénesis Genética , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Heterocromatina/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Mapeo Cromosómico , Metilación de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Eucromatina/química , Eucromatina/metabolismo , Retroalimentación Fisiológica , Frecuencia de los Genes , Haplotipos , Heterocromatina/química , Histonas/genética , Histonas/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Plantas Modificadas Genéticamente , Procesamiento Proteico-Postraduccional , Sitios de Carácter Cuantitativo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
We have studied the three-dimensional (3D) cytoarchitecture of the human hippocampus in neuropathologically healthy and Alzheimer's disease (AD) individuals, based on phase-contrast X-ray computed tomography of postmortem human tissue punch biopsies. In view of recent findings suggesting a nuclear origin of AD, we target in particular the nuclear structure of the dentate gyrus (DG) granule cells. Tissue samples of 20 individuals were scanned and evaluated using a highly automated approach of measurement and analysis, combining multiscale recordings, optimized phase retrieval, segmentation by machine learning, representation of structural properties in a feature space, and classification based on the theory of optimal transport. Accordingly, we find that the prototypical transformation between a structure representing healthy granule cells and the pathological state involves a decrease in the volume of granule cell nuclei, as well as an increase in the electron density and its spatial heterogeneity. The latter can be explained by a higher ratio of heterochromatin to euchromatin. Similarly, many other structural properties can be derived from the data, reflecting both the natural polydispersity of the hippocampal cytoarchitecture between different individuals in the physiological context and the structural effects associated with AD pathology.
Asunto(s)
Mapeo Encefálico/métodos , Hipocampo/diagnóstico por imagen , Imagenología Tridimensional/métodos , Tomografía Computarizada por Rayos X/métodos , Núcleo Celular/metabolismo , Medios de Contraste , Giro Dentado/diagnóstico por imagen , Eucromatina/química , Sustancia Gris/diagnóstico por imagen , Heterocromatina/química , Humanos , Aprendizaje Automático , Distribución Normal , Reconocimiento de Normas Patrones Automatizadas , Análisis de Componente Principal , Reproducibilidad de los Resultados , Sustancia Blanca/diagnóstico por imagenRESUMEN
The SUV39 class of methyltransferase enzymes deposits histone H3 lysine 9 di- and trimethylation (H3K9me2/3), the hallmark of constitutive heterochromatin. How these enzymes are regulated to mark specific genomic regions as heterochromatic is poorly understood. Clr4 is the sole H3K9me2/3 methyltransferase in the fission yeast Schizosaccharomyces pombe, and recent evidence suggests that ubiquitination of lysine 14 on histone H3 (H3K14ub) plays a key role in H3K9 methylation. However, the molecular mechanism of this regulation and its role in heterochromatin formation remain to be determined. Our structure-function approach shows that the H3K14ub substrate binds specifically and tightly to the catalytic domain of Clr4, and thereby stimulates the enzyme by over 250-fold. Mutations that disrupt this mechanism lead to a loss of H3K9me2/3 and abolish heterochromatin silencing similar to clr4 deletion. Comparison with mammalian SET domain proteins suggests that the Clr4 SET domain harbors a conserved sensor for H3K14ub, which mediates licensing of heterochromatin formation.
Asunto(s)
Proteínas de Ciclo Celular , Heterocromatina , Código de Histonas/genética , N-Metiltransferasa de Histona-Lisina , Histonas , Proteínas de Schizosaccharomyces pombe , Dominio Catalítico/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Metilación de ADN/genética , Heterocromatina/química , Heterocromatina/genética , Heterocromatina/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/química , Histonas/genética , Histonas/metabolismo , Lisina/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Ubiquitinación/genéticaRESUMEN
The DNA polymerase zeta (Polζ) plays a critical role in bypassing DNA damage. REV3L, the catalytic subunit of Polζ, is also essential in mouse embryonic development and cell proliferation for reasons that remain incompletely understood. In this study, we reveal that REV3L protein interacts with heterochromatin components including repressive histone marks and localizes in pericentromeric regions through direct interaction with HP1 dimer. We demonstrate that Polζ/REV3L ensures progression of replication forks through difficult-to-replicate pericentromeric heterochromatin, thereby preventing spontaneous chromosome break formation. We also find that Rev3l-deficient cells are compromised in the repair of heterochromatin-associated double-stranded breaks, eliciting deletions in late-replicating regions. Lack of REV3L leads to further consequences that may be ascribed to heterochromatin replication and repair-associated functions of Polζ, with a disruption of the temporal replication program at specific loci. This is correlated with changes in epigenetic landscape and transcriptional control of developmentally regulated genes. These results reveal a new function of Polζ in preventing chromosome instability during replication of heterochromatic regions.
Asunto(s)
Replicación del ADN , Proteínas de Unión al ADN/genética , ADN Polimerasa Dirigida por ADN/genética , ADN/genética , Células Madre Embrionarias/metabolismo , Epigénesis Genética , Heterocromatina/metabolismo , Animales , Línea Celular , Línea Celular Transformada , Proliferación Celular , Homólogo de la Proteína Chromobox 5/genética , Homólogo de la Proteína Chromobox 5/metabolismo , Inestabilidad Cromosómica , ADN/metabolismo , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Embrión de Mamíferos , Células Madre Embrionarias/citología , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células HeLa , Heterocromatina/química , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células 3T3 NIH , Transducción de SeñalRESUMEN
Exposure to the ultraviolet (UV) radiation in sunlight creates DNA lesions, which if left unrepaired can induce mutations and contribute to skin cancer. The two most common UV-induced DNA lesions are the cis-syn cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs), both of which can initiate mutations. Interestingly, mutation frequency across the genomes of many cancers is heterogenous with significant increases in heterochromatin. Corresponding increases in UV lesion susceptibility and decreases in repair are observed in heterochromatin versus euchromatin. However, the individual contributions of CPDs and 6-4PPs to mutagenesis have not been systematically examined in specific genomic and epigenomic contexts. In this study, we compared genome-wide maps of 6-4PP and CPD lesion abundances in primary cells and conducted comprehensive analyses to determine the genetic and epigenetic features associated with susceptibility. Overall, we found a high degree of similarity between 6-4PP and CPD formation, with an enrichment of both in heterochromatin regions. However, when examining the relative levels of the two UV lesions, we found that bivalent and Polycomb-repressed chromatin states were uniquely more susceptible to 6-4PPs. Interestingly, when comparing UV susceptibility and repair with melanoma mutation frequency in these regions, disparate patterns were observed in that susceptibility was not always inversely associated with repair and mutation frequency. Functional enrichment analysis hint at mechanisms of negative selection for these regions that are essential for cell viability, immune function and induce cell death when mutated. Ultimately, these results reveal both the similarities and differences between UV-induced lesions that contribute to melanoma.
Asunto(s)
Reparación del ADN , Epigénesis Genética/efectos de la radiación , Melanoma/genética , Mutación , Neoplasias Cutáneas/genética , Rayos Ultravioleta/efectos adversos , Daño del ADN , Bases de Datos Genéticas , Eucromatina/química , Eucromatina/metabolismo , Eucromatina/efectos de la radiación , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Genoma Humano/efectos de la radiación , Heterocromatina/química , Heterocromatina/metabolismo , Heterocromatina/efectos de la radiación , Histonas/genética , Histonas/metabolismo , Humanos , Melanoma/etiología , Melanoma/metabolismo , Melanoma/patología , Mutagénesis , Proteínas del Grupo Polycomb/genética , Proteínas del Grupo Polycomb/metabolismo , Cultivo Primario de Células , Dímeros de Pirimidina/agonistas , Dímeros de Pirimidina/metabolismo , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patologíaRESUMEN
N6-methyladenine (N6-mA, m6dA, or 6mA), a prevalent DNA modification in prokaryotes, has recently been identified in higher eukaryotes, including mammals. Although 6mA has been well-studied in prokaryotes, the function and regulatory mechanism of 6mA in eukaryotes are still poorly understood. Recent studies indicate that 6mA can serve as an epigenetic mark and play critical roles in various biological processes, from transposable-element suppression to environmental stress response. Here, we review the significant advances in methodology for 6mA detection and major progress in understanding the regulation and function of this non-canonical DNA methylation in eukaryotes, predominantly mammals.
Asunto(s)
Adenina/análogos & derivados , Reparación del ADN , ADN/metabolismo , Epigénesis Genética , Genoma , Adenina/metabolismo , Aminopirina N-Demetilasa/genética , Aminopirina N-Demetilasa/metabolismo , Animales , Cromatografía Líquida de Alta Presión , ADN/genética , Metilación de ADN , Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/metabolismo , Regulación de la Expresión Génica , Heterocromatina/química , Heterocromatina/metabolismo , Humanos , Immunoblotting , Mamíferos , Espectrometría de MasasRESUMEN
BACKGROUND: The origin of sex chromosomes requires the establishment of recombination suppression between the proto-sex chromosomes. In many fish species, the sex chromosome pair is homomorphic with a recent origin, providing species for studying how and why recombination suppression evolved in the initial stages of sex chromosome differentiation, but this requires accurate sequence assembly of the X and Y (or Z and W) chromosomes, which may be difficult if they are recently diverged. RESULTS: Here we produce a haplotype-resolved genome assembly of zig-zag eel (Mastacembelus armatus), an aquaculture fish, at the chromosomal scale. The diploid assembly is nearly gap-free, and in most chromosomes, we resolve the centromeric and subtelomeric heterochromatic sequences. In particular, the Y chromosome, including its highly repetitive short arm, has zero gaps. Using resequencing data, we identify a ~7 Mb fully sex-linked region (SLR), spanning the sex chromosome centromere and almost entirely embedded in the pericentromeric heterochromatin. The SLRs on the X and Y chromosomes are almost identical in sequence and gene content, but both are repetitive and heterochromatic, consistent with zero or low recombination. We further identify an HMG-domain containing gene HMGN6 in the SLR as a candidate sex-determining gene that is expressed at the onset of testis development. CONCLUSIONS: Our study supports the idea that preexisting regions of low recombination, such as pericentromeric regions, can give rise to SLR in the absence of structural variations between the proto-sex chromosomes.
Asunto(s)
Anguilas/genética , Genoma , Proteínas HMGN/genética , Procesos de Determinación del Sexo , Telómero , Cromosoma Y/química , Animales , Centrómero , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Expresión Génica , Proteínas HMGN/metabolismo , Heterocromatina/química , Cariotipo , Masculino , Testículo/crecimiento & desarrollo , Testículo/metabolismo , Cromosoma XRESUMEN
BACKGROUND: Naïve and primed pluripotent stem cells (PSCs) represent two different pluripotent states. Primed PSCs following in vitro culture exhibit lower developmental potency as evidenced by failure in germline chimera assays, unlike mouse naïve PSCs. However, the molecular mechanisms underlying the lower developmental competency of primed PSCs remain elusive. RESULTS: We examine the regulation of telomere maintenance, retrotransposon activity, and genomic stability of primed PSCs and compare them with naïve PSCs. Surprisingly, primed PSCs only minimally maintain telomeres and show fragile telomeres, associated with declined DNA recombination and repair activity, in contrast to naïve PSCs that robustly elongate telomeres. Also, we identify LINE1 family integrant L1Md_T as naïve-specific retrotransposon and ERVK family integrant IAPEz to define primed PSCs, and their transcription is differentially regulated by heterochromatic histones and Dnmt3b. Notably, genomic instability of primed PSCs is increased, in association with aberrant retrotransposon activity. CONCLUSIONS: Our data suggest that fragile telomere, retrotransposon-associated genomic instability, and declined DNA recombination repair, together with reduced function of cell cycle and mitochondria, increased apoptosis, and differentiation properties may link to compromised developmental potency of primed PSCs, noticeably distinguishable from naïve PSCs.
Asunto(s)
Inestabilidad Genómica , Células Madre Pluripotentes/metabolismo , Procesamiento Proteico-Postraduccional , Retroelementos , Homeostasis del Telómero , Activinas/farmacología , Animales , Apoptosis/genética , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Diferenciación Celular/efectos de los fármacos , ADN/genética , ADN/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Factor 2 de Crecimiento de Fibroblastos/farmacología , Heterocromatina/química , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Endogámicos ICR , Mitocondrias/genética , Mitocondrias/metabolismo , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/efectos de los fármacos , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Reparación del ADN por Recombinación , Telómero/metabolismo , Telómero/ultraestructura , ADN Metiltransferasa 3BRESUMEN
Small non-protein coding RNAs are involved in pathways that control the genome at the level of chromatin. In Schizosaccharomyces pombe, small interfering RNAs (siRNAs) are required for the faithful propagation of heterochromatin that is found at peri-centromeric repeats. In contrast to repetitive DNA, protein-coding genes are refractory to siRNA-mediated heterochromatin formation, unless siRNAs are expressed in mutant cells. Here we report the identification of 20 novel mutant alleles that enable de novo formation of heterochromatin at a euchromatic protein-coding gene by using trans-acting siRNAs as triggers. For example, a single amino acid substitution in the pre-mRNA cleavage factor Yth1 enables siRNAs to trigger silent chromatin formation with unparalleled efficiency. Our results are consistent with a kinetic nascent transcript processing model for the inhibition of small-RNA-directed de novo formation of heterochromatin and lay a foundation for further mechanistic dissection of cellular activities that counteract epigenetic gene silencing.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Silenciador del Gen , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Schizosaccharomyces/genética , Factores de Escisión y Poliadenilación de ARNm/genética , Alelos , Sustitución de Aminoácidos , Centrómero/química , Centrómero/metabolismo , Ensamble y Desensamble de Cromatina , Perfilación de la Expresión Génica , Heterocromatina/química , Heterocromatina/metabolismo , Cinética , Modelos Genéticos , Anotación de Secuencia Molecular , Mutación , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Schizosaccharomyces/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
Pericentromeric heterochromatin (PCH), the constitutive heterochromatin of pericentromeric regions, plays crucial roles in various cellular events, such as cell division and DNA replication. PCH forms chromocenters in the interphase nucleus, and chromocenters cluster at the prophase of meiosis. Chromocenter clustering has been reported to be critical for the appropriate progression of meiosis. However, the molecular mechanisms underlying chromocenter clustering remain elusive. In this study, we found that global DNA hypomethylation, 5hmC enrichment in PCH, and chromocenter clustering of Dnmt1-KO ESCs were similar to those of the female meiotic germ cells. Tet1 is essential for the deposition of 5hmC and facultative histone marks of H3K27me3 and H2AK119ub at PCH, as well as chromocenter clustering. RING1B, one of the core components of PRC1, is recruited to PCH by TET1, and PRC1 plays a critical role in chromocenter clustering. In addition, the rearrangement of the chromocenter under DNA hypomethylated condition was mediated by liquid-liquid phase separation. Thus, we demonstrated a novel role of Tet1 in chromocenter rearrangement in DNA hypomethylated cells.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasa 1/genética , Proteínas de Unión al ADN/genética , ADN/genética , Epigénesis Genética , Heterocromatina/química , Células Madre Embrionarias de Ratones/metabolismo , Proteínas Proto-Oncogénicas/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animales , Línea Celular , Centrómero/química , Centrómero/metabolismo , ADN/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/deficiencia , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Femenino , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Meiosis , Ratones , Células Madre Embrionarias de Ratones/citología , Óvulo/citología , Óvulo/metabolismo , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Liquid-liquid phase separation (LLPS) of proteins and DNA has recently emerged as a possible mechanism underlying the dynamic organization of chromatin. We herein report the role of DNA quadruplex folding in liquid droplet formation via LLPS induced by interactions between DNA and linker histone H1 (H1), a key regulator of chromatin organization. Fluidity measurements inside the droplets, binding assays using G-quadruplex-selective probes, and structural analyses based on circular dichroism demonstrated that quadruplex DNA structures, such as the G-quadruplex and i-motif, promote droplet formation with H1 and decrease molecular motility within droplets. The dissolution of the droplets in the presence of additives and the LLPS of the DNA structural units indicated that, in addition to electrostatic interactions between the DNA and the intrinsically disordered region of H1, π-π stacking between quadruplex DNAs could potentially drive droplet formation, unlike in the electrostatically driven LLPS of duplex DNA and H1. According to phase diagrams of anionic molecules with various conformations, the high LLPS ability associated with quadruplex folding arises from the formation of interfaces consisting of organized planes of guanine bases and the side surfaces with a high charge density. Given that DNA quadruplex structures are well-documented in heterochromatin regions, it is imperative to understand the role of DNA quadruplex folding in the context of intranuclear LLPS.
Asunto(s)
ADN/química , Histonas/química , Secuencia de Aminoácidos , G-Cuádruplex , Heterocromatina/química , Extracción Líquido-Líquido , Unión Proteica , Dominios ProteicosRESUMEN
Selection of C-terminal motifs participated in evolution of distinct histone H2A variants. Hybrid types of variants combining motifs from distinct H2A classes are extremely rare. This suggests that the proximity between the motif cases interferes with their function. We studied this question in flowering plants that evolved sporadically a hybrid H2A variant combining the SQ motif of H2A.X that participates in the DNA damage response with the KSPK motif of H2A.W that stabilizes heterochromatin. Our inventory of PTMs of H2A.W variants showed that in vivo the cell cycle-dependent kinase CDKA phosphorylates the KSPK motif of H2A.W but only in absence of an SQ motif. Phosphomimicry of KSPK prevented DNA damage response by the SQ motif of the hybrid H2A.W/X variant. In a synthetic yeast expressing the hybrid H2A.W/X variant, phosphorylation of KSPK prevented binding of the BRCT-domain protein Mdb1 to phosphorylated SQ and impaired response to DNA damage. Our findings illustrate that PTMs mediate interference between the function of H2A variant specific C-terminal motifs. Such interference could explain the mutual exclusion of motifs that led to evolution of H2A variants.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Histonas/genética , Procesamiento Proteico-Postraduccional , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Técnicas de Cultivo de Célula , Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Daño del ADN , Evolución Molecular , Heterocromatina/química , Heterocromatina/metabolismo , Histonas/metabolismo , Fosforilación , Células Vegetales/metabolismo , Plantas Modificadas Genéticamente , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , TransgenesRESUMEN
Heterochromatin is a key architectural feature of eukaryotic genomes crucial for silencing of repetitive elements. During Drosophila embryonic cellularization, heterochromatin rapidly appears over repetitive sequences, but the molecular details of how heterochromatin is established are poorly understood. Here, we map the genome-wide distribution of H3K9me3-dependent heterochromatin in individual embryos of Drosophila miranda at precisely staged developmental time points. We find that canonical H3K9me3 enrichment is established prior to cellularization and matures into stable and broad heterochromatin domains through development. Intriguingly, initial nucleation sites of H3K9me3 enrichment appear as early as embryonic stage 3 over transposable elements (TEs) and progressively broaden, consistent with spreading to neighboring nucleosomes. The earliest nucleation sites are limited to specific regions of a small number of recently active retrotransposon families and often appear over promoter and 5' regions of LTR retrotransposons, while late nucleation sites develop broadly across the entirety of most TEs. Interestingly, early nucleating TEs are strongly associated with abundant maternal piRNAs and show early zygotic transcription. These results support a model of piRNA-associated co-transcriptional silencing while also suggesting additional mechanisms for site-restricted H3K9me3 nucleation at TEs in pre-cellular Drosophila embryos.