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1.
Elife ; 112022 01 04.
Artículo en Inglés | MEDLINE | ID: mdl-34982026

RESUMEN

Vertebrate embryos achieve developmental competency during zygotic genome activation (ZGA) by establishing chromatin states that silence yet poise developmental genes for subsequent lineage-specific activation. Here, we reveal the order of chromatin states in establishing developmental gene poising in preZGA zebrafish embryos. Poising is established at promoters and enhancers that initially contain open/permissive chromatin with 'Placeholder' nucleosomes (bearing H2A.Z, H3K4me1, and H3K27ac), and DNA hypomethylation. Silencing is initiated by the recruitment of polycomb repressive complex 1 (PRC1), and H2Aub1 deposition by catalytic Rnf2 during preZGA and ZGA stages. During postZGA, H2Aub1 enables Aebp2-containing PRC2 recruitment and H3K27me3 deposition. Notably, preventing H2Aub1 (via Rnf2 inhibition) eliminates recruitment of Aebp2-PRC2 and H3K27me3, and elicits transcriptional upregulation of certain developmental genes during ZGA. However, upregulation is independent of H3K27me3 - establishing H2Aub1 as the critical silencing modification at ZGA. Taken together, we reveal the logic and mechanism for establishing poised/silent developmental genes in early vertebrate embryos.


Asunto(s)
Cromatina/genética , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Proteínas del Grupo Polycomb/metabolismo , Pez Cebra/genética , Cigoto/crecimiento & desarrollo , Animales , Línea Celular , Metilación de ADN , Drosophila , Proteínas de Drosophila/genética , Genes del Desarrollo , Genómica/métodos , Proteínas del Grupo Polycomb/genética , Cigoto/metabolismo
2.
Genome Res ; 31(6): 981-994, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-34006569

RESUMEN

Chromatin architecture mapping in 3D formats has increased our understanding of how regulatory sequences and gene expression are connected and regulated in a genome. The 3D chromatin genome shows extensive remodeling during embryonic development, and although the cleavage-stage embryos of most species lack structure before zygotic genome activation (pre-ZGA), zebrafish has been reported to have structure. Here, we aimed to determine the chromosomal architecture in paternal/sperm zebrafish gamete cells to discern whether it either resembles or informs early pre-ZGA zebrafish embryo chromatin architecture. First, we assessed the higher-order architecture through advanced low-cell in situ Hi-C. The structure of zebrafish sperm, packaged by histones, lacks topological associated domains and instead displays "hinge-like" domains of ∼150 kb that repeat every 1-2 Mbs, suggesting a condensed repeating structure resembling mitotic chromosomes. The pre-ZGA embryos lacked chromosomal structure, in contrast to prior work, and only developed structure post-ZGA. During post-ZGA, we find chromatin architecture beginning to form at small contact domains of a median length of ∼90 kb. These small contact domains are established at enhancers, including super-enhancers, and chemical inhibition of Ep300a (p300) and Crebbpa (CBP) activity, lowering histone H3K27ac, but not transcription inhibition, diminishes these contacts. Together, this study reveals hinge-like domains in histone-packaged zebrafish sperm chromatin and determines that the initial formation of high-order chromatin architecture in zebrafish embryos occurs after ZGA primarily at enhancers bearing high H3K27ac.


Asunto(s)
Cromatina , Pez Cebra , Animales , Cromatina/genética , Cromatina/metabolismo , Cromosomas/genética , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Masculino , Espermatozoides/metabolismo , Pez Cebra/genética , Cigoto
3.
Genome Res ; 31(6): 968-980, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-34006570

RESUMEN

Chromatin looping plays an important role in genome regulation. However, because ChIP-seq and loop-resolution Hi-C (DNA-DNA proximity ligation) are extremely challenging in mammalian early embryos, the developmental stage at which cohesin-mediated loops form remains unknown. Here, we study early development in medaka (the Japanese killifish, Oryzias latipes) at 12 time points before, during, and after gastrulation (the onset of cell differentiation) and characterize transcription, protein binding, and genome architecture. We find that gastrulation is associated with drastic changes in genome architecture, including the formation of the first loops between sites bound by the insulator protein CTCF and a large increase in the size of contact domains. In contrast, the binding of the CTCF is fixed throughout embryogenesis. Loops form long after genome-wide transcriptional activation, and long after domain formation seen in mouse embryos. These results suggest that, although loops may play a role in differentiation, they are not required for zygotic transcription. When we repeated our experiments in zebrafish, loops did not emerge until gastrulation, that is, well after zygotic genome activation. We observe that loop positions are highly conserved in synteny blocks of medaka and zebrafish, indicating that the 3D genome architecture has been maintained for >110-200 million years of evolution.


Asunto(s)
Oryzias , Animales , Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Proteínas de Ciclo Celular/genética , Cromatina/genética , Gastrulación/genética , Ratones , Oryzias/genética , Pez Cebra/genética
4.
Cell ; 172(5): 993-1006.e13, 2018 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-29456083

RESUMEN

The fate and function of epigenetic marks during the germline-to-embryo transition is a key issue in developmental biology, with relevance to stem cell programming and transgenerational inheritance. In zebrafish, DNA methylation patterns are programmed in transcriptionally quiescent cleavage embryos; paternally inherited patterns are maintained, whereas maternal patterns are reprogrammed to match the paternal. Here, we provide the mechanism by demonstrating that "Placeholder" nucleosomes, containing histone H2A variant H2A.Z(FV) and H3K4me1, virtually occupy all regions lacking DNA methylation in both sperm and cleavage embryos and reside at promoters encoding housekeeping and early embryonic transcription factors. Upon genome-wide transcriptional onset, genes with Placeholder become either active (H3K4me3) or silent (H3K4me3/K27me3). Notably, perturbations causing Placeholder loss confer DNA methylation accumulation, whereas acquisition/expansion of Placeholder confers DNA hypomethylation and improper gene activation. Thus, during transcriptionally quiescent gametic and embryonic stages, an H2A.Z(FV)/H3K4me1-containing Placeholder nucleosome deters DNA methylation, poising parental genes for either gene-specific activation or facultative repression.


Asunto(s)
Reprogramación Celular/genética , Metilación de ADN/genética , Embrión no Mamífero/metabolismo , Células Germinativas/metabolismo , Nucleosomas/metabolismo , Animales , Histonas/metabolismo , Masculino , Mutación/genética , Espermatozoides/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
5.
Cell Stem Cell ; 21(4): 533-546.e6, 2017 10 05.
Artículo en Inglés | MEDLINE | ID: mdl-28985528

RESUMEN

Human adult spermatogonial stem cells (hSSCs) must balance self-renewal and differentiation. To understand how this is achieved, we profiled DNA methylation and open chromatin (ATAC-seq) in SSEA4+ hSSCs, analyzed bulk and single-cell RNA transcriptomes (RNA-seq) in SSEA4+ hSSCs and differentiating c-KIT+ spermatogonia, and performed validation studies via immunofluorescence. First, DNA hypomethylation at embryonic developmental genes supports their epigenetic "poising" in hSSCs for future/embryonic expression, while core pluripotency genes (OCT4 and NANOG) were transcriptionally and epigenetically repressed. Interestingly, open chromatin in hSSCs was strikingly enriched in binding sites for pioneer factors (NFYA/B, DMRT1, and hormone receptors). Remarkably, single-cell RNA-seq clustering analysis identified four cellular/developmental states during hSSC differentiation, involving major transitions in cell-cycle and transcriptional regulators, splicing and signaling factors, and glucose/mitochondria regulators. Overall, our results outline the dynamic chromatin/transcription landscape operating in hSSCs and identify crucial molecular pathways that accompany the transition from quiescence to proliferation and differentiation.


Asunto(s)
Cromatina/metabolismo , Análisis de Secuencia de ARN/métodos , Transducción de Señal , Análisis de la Célula Individual/métodos , Espermatogonias/citología , Células Madre/citología , Células Madre/metabolismo , Secuencia de Bases , Sitios de Unión , Análisis por Conglomerados , ADN/metabolismo , Metilación de ADN/genética , Genómica , Humanos , Masculino , Meiosis , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Receptores de Superficie Celular/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos/genética , Reproducibilidad de los Resultados , Túbulos Seminíferos/citología , Antígenos Embrionarios Específico de Estadio/metabolismo , Transcripción Genética , Transcriptoma/genética
6.
Nat Genet ; 49(6): 925-934, 2017 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-28459457

RESUMEN

To better understand transcriptional regulation during human oogenesis and preimplantation development, we defined stage-specific transcription, which highlighted the cleavage stage as being highly distinctive. Here, we present multiple lines of evidence that a eutherian-specific multicopy retrogene, DUX4, encodes a transcription factor that activates hundreds of endogenous genes (for example, ZSCAN4, KDM4E and PRAMEF-family genes) and retroviral elements (MERVL/HERVL family) that define the cleavage-specific transcriptional programs in humans and mice. Remarkably, mouse Dux expression is both necessary and sufficient to convert mouse embryonic stem cells (mESCs) into 2-cell-embryo-like ('2C-like') cells, measured here by the reactivation of '2C' genes and repeat elements, the loss of POU5F1 (also known as OCT4) protein and chromocenters, and the conversion of the chromatin landscape (as assessed by transposase-accessible chromatin using sequencing (ATAC-seq)) to a state strongly resembling that of mouse 2C embryos. Thus, we propose mouse DUX and human DUX4 as major drivers of the cleavage or 2C state.


Asunto(s)
Proteínas de Homeodominio/metabolismo , Retroelementos/genética , Adulto , Empalme Alternativo , Animales , Blastocisto/fisiología , Cromatina/genética , Cromatina/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Ratones Transgénicos , Oocitos/fisiología , Transcriptoma
7.
Cell Cycle ; 15(16): 2216-2225, 2016 Aug 17.
Artículo en Inglés | MEDLINE | ID: mdl-27248858

RESUMEN

The cell tightly controls histone protein levels in order to achieve proper packaging of the genome into chromatin, while avoiding the deleterious consequences of excess free histones. Our accompanying study has shown that a histone modification that loosens the intrinsic structure of the nucleosome, phosphorylation of histone H3 on threonine 118 (H3 T118ph), exists on centromeres and chromosome arms during mitosis. Here, we show that H3 T118ph localizes to centrosomes in humans, flies, and worms during all stages of mitosis. H3 abundance at the centrosome increased upon proteasome inhibition, suggesting that excess free histone H3 localizes to centrosomes for degradation during mitosis. In agreement, we find ubiquitinated H3 specifically during mitosis and within purified centrosomes. These results suggest that targeting of histone H3 to the centrosome for proteasome-mediated degradation is a novel pathway for controlling histone supply, specifically during mitosis.


Asunto(s)
Centrosoma/metabolismo , Histonas/metabolismo , Mitosis , Proteolisis , Animales , Proteínas de Ciclo Celular/metabolismo , Centrosoma/efectos de los fármacos , Células HeLa , Humanos , Mitosis/efectos de los fármacos , Modelos Biológicos , Mutación/genética , Inhibidores de Proteasoma/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteolisis/efectos de los fármacos , Proteínas Proto-Oncogénicas/metabolismo , Ubiquitina/metabolismo , Ubiquitinación , Quinasa Tipo Polo 1
8.
Elife ; 5: e11402, 2016 Feb 16.
Artículo en Inglés | MEDLINE | ID: mdl-26878753

RESUMEN

Phosphorylation of histone H3 threonine 118 (H3 T118ph) weakens histone DNA-contacts, disrupting the nucleosome structure. We show that Aurora-A mediated H3 T118ph occurs at pericentromeres and chromosome arms during prophase and is lost upon chromosome alignment. Expression of H3 T118E or H3 T118I (a SIN mutation that bypasses the need for the ATP-dependent nucleosome remodeler SWI/SNF) leads to mitotic problems including defects in spindle attachment, delayed cytokinesis, reduced chromatin packaging, cohesion loss, cohesin and condensin I loss in human cells. In agreement, overexpression of Aurora-A leads to increased H3 T118ph levels, causing cohesion loss, and reduced levels of cohesin and condensin I on chromatin. Normal levels of H3 T118ph are important because it is required for development in fruit flies. We propose that H3 T118ph alters the chromatin structure during specific phases of mitosis to promote timely condensin I and cohesin disassociation, which is essential for effective chromosome segregation.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Aurora Quinasa A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Histonas/metabolismo , Mitosis , Complejos Multiproteicos/metabolismo , Procesamiento Proteico-Postraduccional , Treonina/metabolismo , Animales , Línea Celular , ADN/metabolismo , Drosophila , Humanos , Fosforilación , Cohesinas
9.
Mol Biosyst ; 4(6): 663-71, 2008 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-18493665

RESUMEN

Mycolactone is a polyketide natural product secreted by Mycobacterium ulcerans, the organism responsible for the tropical skin disease Buruli ulcer. The finding that this small molecule virulence factor is sufficient to reconstitute the necrotic pathology associated with Buruli ulcer suggests that a better understanding of mycolactone biosynthesis, particularly the processes which are distinct from those in human metabolism, may provide a unique avenue for the development of selective therapeutics. In the present study we have cloned, expressed, and biochemically characterized the putative macrocycle forming thioesterase for mycolactone, MLSA2 TE. We have evaluated the enzyme both as the truncated thioesterase domain and as a carrier protein-linked didomain construct. The results of these analyses distinguish MLSA2 TE from traditional fatty acid and polyketide synthase TE-domains in terms of its sequence, kinetic parameters, and susceptibility to traditional active-site directed inhibitors. These findings suggest that MLSA2 TE utilizes a unique biochemical mechanism for macrocycle formation.


Asunto(s)
Toxinas Bacterianas/metabolismo , Compuestos Macrocíclicos/metabolismo , Tioléster Hidrolasas/metabolismo , Secuencia de Aminoácidos , Toxinas Bacterianas/química , Catálisis , Dominio Catalítico , Clonación Molecular , Ciclización , Perfilación de la Expresión Génica , Hidrólisis , Cinética , Compuestos Macrocíclicos/química , Macrólidos , Conformación Molecular , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Análisis de Secuencia , Tioléster Hidrolasas/genética , Tioléster Hidrolasas/aislamiento & purificación
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