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1.
Science ; 372(6537): 91-94, 2021 04 02.
Artículo en Inglés | MEDLINE | ID: mdl-33795458

RESUMEN

Neurons are the longest-lived cells in our bodies and lack DNA replication, which makes them reliant on a limited repertoire of DNA repair mechanisms to maintain genome fidelity. These repair mechanisms decline with age, but we have limited knowledge of how genome instability emerges and what strategies neurons and other long-lived cells may have evolved to protect their genomes over the human life span. A targeted sequencing approach in human embryonic stem cell-induced neurons shows that, in neurons, DNA repair is enriched at well-defined hotspots that protect essential genes. These hotspots are enriched with histone H2A isoforms and RNA binding proteins and are associated with evolutionarily conserved elements of the human genome. These findings provide a basis for understanding genome integrity as it relates to aging and disease in the nervous system.


Asunto(s)
Reparación del ADN , Genoma Humano , Inestabilidad Genómica , Neuronas/metabolismo , Envejecimiento/genética , Daño del ADN , ADN Intergénico , Desoxiuridina/análogos & derivados , Desoxiuridina/metabolismo , Células Madre Embrionarias , Histonas/metabolismo , Humanos , Mitosis , Mutación , Enfermedades del Sistema Nervioso/genética , Neuronas/citología , Regiones Promotoras Genéticas , Proteínas de Unión al ARN/metabolismo , Análisis de Secuencia de ADN , Transcripción Genética
2.
Nat Genet ; 53(4): 539-550, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33821003

RESUMEN

Parental epigenomes are established during gametogenesis. While they are largely reset after fertilization, broad domains of Polycomb repressive complex 2 (PRC2)-mediated formation of lysine 27-trimethylated histone H3 (H3K27me3) are inherited from oocytes in mice. How maternal H3K27me3 is established and inherited by embryos remains elusive. Here, we show that PRC1-mediated formation of lysine 119-monoubiquititinated histone H2A (H2AK119ub1) confers maternally heritable H3K27me3. Temporal profiling of H2AK119ub1 dynamics revealed that atypically broad H2AK119ub1 domains are established, along with H3K27me3, during oocyte growth. From the two-cell stage, H2AK119ub1 is progressively deposited at typical Polycomb targets and precedes H3K27me3. Reduction of H2AK119ub1 by depletion of Polycomb group ring finger 1 (PCGF1) and PCGF6-essential components of variant PRC1 (vPRC1)-leads to H3K27me3 loss at a subset of genes in oocytes. The gene-selective H3K27me3 deficiency is irreversibly inherited by embryos, causing loss of maternal H3K27me3-dependent imprinting, embryonic sublethality and placental enlargement at term. Collectively, our study unveils preceding dynamics of H2AK119ub1 over H3K27me3 at the maternal-to-zygotic transition, and identifies PCGF1/6-vPRC1 as an essential player in maternal epigenetic inheritance.


Asunto(s)
Embrión de Mamíferos/metabolismo , Epigénesis Genética , Histonas/genética , Herencia Materna , Complejo Represivo Polycomb 1/genética , Animales , Embrión de Mamíferos/citología , Epigenoma , Femenino , Fertilización/genética , Histonas/metabolismo , Lisina/metabolismo , Masculino , Ratones , Oocitos/citología , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Embarazo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Espermatozoides/citología , Espermatozoides/metabolismo , Ubiquitinación , Cigoto/citología , Cigoto/crecimiento & desarrollo , Cigoto/metabolismo
3.
Nat Genet ; 53(4): 551-563, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33821005

RESUMEN

Polycomb repressive complexes 1 and 2 (PRC1/2) maintain transcriptional silencing of developmental genes largely by catalyzing the formation of mono-ubiquitinated histone H2A at lysine 119 (H2AK119ub1) and trimethylated histone H3 at lysine 27 (H3K27me3), respectively. How Polycomb domains are reprogrammed during mammalian preimplantation development remains largely unclear. Here we show that, although H2AK119ub1 and H3K27me3 are highly colocalized in gametes, they undergo differential reprogramming dynamics following fertilization. H3K27me3 maintains thousands of maternally biased domains until the blastocyst stage, whereas maternally biased H2AK119ub1 distribution in zygotes is largely equalized at the two-cell stage. Notably, while maternal PRC2 depletion has a limited effect on global H2AK119ub1 in early embryos, it disrupts allelic H2AK119ub1 at H3K27me3 imprinting loci including Xist. By contrast, acute H2AK119ub1 depletion in zygotes does not affect H3K27me3 imprinting maintenance, at least by the four-cell stage. Importantly, loss of H2AK119ub1, but not H3K27me3, causes premature activation of developmental genes during zygotic genome activation (ZGA) and subsequent embryonic arrest. Thus, our study reveals distinct dynamics and functions of H3K27me3 and H2AK119ub1 in mouse preimplantation embryos.


Asunto(s)
Blastocisto/metabolismo , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Histonas/genética , Herencia Materna , ARN Largo no Codificante/genética , Animales , Blastocisto/citología , Femenino , Fertilización/genética , Histonas/metabolismo , Lisina/metabolismo , Masculino , Ratones , Oocitos/citología , Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Herencia Paterna , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Embarazo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Largo no Codificante/metabolismo , Espermatozoides/citología , Espermatozoides/metabolismo , Ubiquitinación , Cigoto/citología , Cigoto/crecimiento & desarrollo , Cigoto/metabolismo
4.
Nat Genet ; 53(4): 487-499, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33795866

RESUMEN

The relationship between chromatin organization and gene regulation remains unclear. While disruption of chromatin domains and domain boundaries can lead to misexpression of developmental genes, acute depletion of regulators of genome organization has a relatively small effect on gene expression. It is therefore uncertain whether gene expression and chromatin state drive chromatin organization or whether changes in chromatin organization facilitate cell-type-specific activation of gene expression. Here, using the dorsoventral patterning of the Drosophila melanogaster embryo as a model system, we provide evidence for the independence of chromatin organization and dorsoventral gene expression. We define tissue-specific enhancers and link them to expression patterns using single-cell RNA-seq. Surprisingly, despite tissue-specific chromatin states and gene expression, chromatin organization is largely maintained across tissues. Our results indicate that tissue-specific chromatin conformation is not necessary for tissue-specific gene expression but rather acts as a scaffold facilitating gene expression when enhancers become active.


Asunto(s)
Tipificación del Cuerpo/genética , Linaje de la Célula/genética , Cromatina/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Animales , Animales Modificados Genéticamente , Diferenciación Celular , Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/metabolismo , Embrión no Mamífero , Elementos de Facilitación Genéticos , Femenino , Genoma , Secuenciación de Nucleótidos de Alto Rendimiento , Histonas/genética , Histonas/metabolismo , Masculino , Especificidad de Órganos , Regiones Promotoras Genéticas , Análisis de la Célula Individual , Transcripción Genética
5.
Int J Mol Sci ; 22(7)2021 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-33810274

RESUMEN

The carbazole compounds PK9320 (1-(9-ethyl-7-(furan-2-yl)-9H-carbazol-3-yl)-N-methylmethanamine) and PK9323 (1-(9-ethyl-7-(thiazol-4-yl)-9H-carbazol-3-yl)-N-methylmethanamine), second-generation analogues of PK083 (1-(9-ethyl-9H-carbazol-3-yl)-N-methylmethanamine), restore p53 signaling in Y220C p53-mutated cancer cells by binding to a mutation-induced surface crevice and acting as molecular chaperones. In the present paper, these three molecules have been tested for mutant p53-independent genotoxic and epigenomic effects on wild-type p53 MCF-7 breast adenocarcinoma cells, employing a combination of Western blot for phospho-γH2AX histone, Comet assay and methylation-sensitive arbitrarily primed PCR to analyze their intrinsic DNA damage-inducing and DNA methylation-changing abilities. We demonstrate that small modifications in the substitution patterns of carbazoles can have profound effects on their intrinsic genotoxic and epigenetic properties, with PK9320 and PK9323 being eligible candidates as "anticancer compounds" and "anticancer epi-compounds" and PK083 a "damage-corrective" compound on human breast adenocarcinoma cells. Such different properties may be exploited for their use as anticancer agents and chemical probes.


Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/metabolismo , Carbazoles/farmacología , Mutágenos/farmacología , Antineoplásicos/química , Neoplasias de la Mama/genética , Carbazoles/química , Daño del ADN , Metilación de ADN , Epigénesis Genética/efectos de los fármacos , Femenino , Histonas/metabolismo , Humanos , Células MCF-7 , Mutágenos/química , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo
6.
Int J Mol Sci ; 22(7)2021 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-33810299

RESUMEN

Rosuvastatin (RST) is primarily used to treat high cholesterol levels. As it has potentially harmful but not well-documented effects on embryos, RST is contraindicated during pregnancy. To demonstrate whether RST could induce molecular epigenetic events in the brains of newborn rats, pregnant mothers were treated daily with oral RST from the 11th day of pregnancy for 10 days (or until delivery). On postnatal day 1, the brains of the control and RST-treated rats were removed for Western blot or immunohistochemical analyses. Several antibodies that recognize different methylation sites for H2A, H2B, H3, and H4 histones were quantified. Analyses of cell-type-specific markers in the newborn brains demonstrated that prenatal RST administration did not affect the composition and cell type ratios as compared to the controls. Prenatal RST administration did, however, induce a general, nonsignificant increase in H2AK118me1, H2BK5me1, H3, H3K9me3, H3K27me3, H3K36me2, H4, H4K20me2, and H4K20me3 levels, compared to the controls. Moreover, significant changes were detected in the number of H3K4me1 and H3K4me3 sites (134.3% ± 19.2% and 127.8% ± 8.5% of the controls, respectively), which are generally recognized as transcriptional activators. Fluorescent/confocal immunohistochemistry for cell-type-specific markers and histone methylation marks on tissue sections indicated that most of the increase at these sites belonged to neuronal cell nuclei. Thus, prenatal RST treatment induces epigenetic changes that could affect neuronal differentiation and development.


Asunto(s)
Anticolesterolemiantes/efectos adversos , Encéfalo/efectos de los fármacos , Embrión de Mamíferos/efectos de los fármacos , Epigénesis Genética , Código de Histonas , Rosuvastatina Cálcica/efectos adversos , Animales , Anticolesterolemiantes/farmacología , Encéfalo/embriología , Encéfalo/metabolismo , Femenino , Histonas/efectos de los fármacos , Histonas/metabolismo , Metilación , Ratas , Ratas Sprague-Dawley , Rosuvastatina Cálcica/farmacología
7.
Methods Mol Biol ; 2269: 37-47, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33687670

RESUMEN

Ionizing radiation is a critical component of glioblastoma (GBM) therapy. Recent data have implicated glioblastoma stem-like cells (GSCs) as determinants of GBM development, maintenance, and treatment response. Understanding the response of GSCs to radiation should thus provide insight into the development of improved GBM treatment strategies. Towards this end, in vitro techniques for the analysis of GSC radiosensitivity are an essential starting point. One such method, the clonogenic survival assay has been adapted to assessing the intrinsic radiosensitivity of GSCs and is described here. As an alternative method, the limiting dilution assay is presented for defining the radiosensitivity of GSC lines that do not form colonies or only grow as neurospheres. In addition to these cellular strategies, we describe γH2AX foci analysis, which provides a surrogate marker for radiosensitivity at the molecular level. Taken together, the in vitro methods presented here provide tools for defining intrinsic radiosensitivity of GSCs and for testing agents that may enhance GBM radioresponse.


Asunto(s)
Biomarcadores de Tumor , Sitios Genéticos , Glioblastoma , Histonas , Proteínas de Neoplasias , Células Madre Neoplásicas , Tolerancia a Radiación , Biomarcadores de Tumor/metabolismo , Línea Celular Tumoral , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Glioblastoma/radioterapia , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Esferoides Celulares/metabolismo , Esferoides Celulares/patología
8.
Nat Commun ; 12(1): 1630, 2021 03 12.
Artículo en Inglés | MEDLINE | ID: mdl-33712604

RESUMEN

Cell type-specific enhancers are activated by coordinated actions of lineage-determining transcription factors (LDTFs) and chromatin regulators. The SWI/SNF chromatin remodeling complex BAF and the histone H3K4 methyltransferase MLL4 (KMT2D) are both implicated in enhancer activation. However, the interplay between BAF and MLL4 in enhancer activation remains unclear. Using adipogenesis as a model system, we identify BAF as the major SWI/SNF complex that colocalizes with MLL4 and LDTFs on active enhancers and is required for cell differentiation. In contrast, the promoter enriched SWI/SNF complex PBAF is dispensable for adipogenesis. By depleting BAF subunits SMARCA4 (BRG1) and SMARCB1 (SNF5) as well as MLL4 in cells, we show that BAF and MLL4 reciprocally regulate each other's binding on active enhancers before and during adipogenesis. By focusing on enhancer activation by the adipogenic pioneer transcription factor C/EBPß without inducing cell differentiation, we provide direct evidence for an interdependent relationship between BAF and MLL4 in activating cell type-specific enhancers. Together, these findings reveal a positive feedback between BAF and MLL4 in promoting LDTF-dependent activation of cell type-specific enhancers.


Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Elementos de Facilitación Genéticos , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Adipogénesis/genética , Animales , Diferenciación Celular , Cromatina , Ensamble y Desensamble de Cromatina , ADN Helicasas , Regulación de la Expresión Génica , Histonas/metabolismo , Ratones , Proteínas Nucleares , Regiones Promotoras Genéticas , Proteína SMARCB1 , Factores de Transcripción
9.
10.
J Vis Exp ; (169)2021 03 04.
Artículo en Inglés | MEDLINE | ID: mdl-33749685

RESUMEN

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.


Asunto(s)
Biomarcadores/metabolismo , Epigénesis Genética , Histonas/aislamiento & purificación , Espectrometría de Masas , Hojas de la Planta/metabolismo , Proteínas de Plantas/aislamiento & purificación , Sorghum/genética , Sorghum/metabolismo , Secuencia de Aminoácidos , Tampones (Química) , Núcleo Celular/metabolismo , Cromatografía Liquida , Histonas/química , Histonas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Procesamiento Proteico-Postraduccional
11.
Nat Commun ; 12(1): 1828, 2021 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-33758195

RESUMEN

DNA sequences containing consecutive guanines organized in 4-interspaced tandem repeats can form stable single-stranded secondary structures, called G-quadruplexes (G4). Herein, we report that the Polycomb group protein BMI1 is enriched at heterochromatin regions containing putative G4 DNA sequences, and that G4 structures accumulate in cells with reduced BMI1 expression and/or relaxed chromatin, including sporadic Alzheimer's disease (AD) neurons. In AD neurons, G4 structures preferentially accumulate in lamina-associated domains, and this is rescued by re-establishing chromatin compaction. ChIP-seq analyses reveal that G4 peaks correspond to evolutionary conserved Long Interspersed Element-1 (L1) sequences predicted to be transcriptionally active. Hence, G4 structures co-localize with RNAPII, and inhibition of transcription can reverse the G4 phenotype without affecting chromatin's state, thus uncoupling both components. Intragenic G4 structures affecting splicing events are furthermore associated with reduced neuronal gene expression in AD. Active L1 sequences are thus at the origin of most G4 structures observed in human neurons.


Asunto(s)
Enfermedad de Alzheimer/metabolismo , Eucromatina/metabolismo , G-Cuádruplex , Elementos de Nucleótido Esparcido Largo/genética , Neurogénesis/genética , Neuronas/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Enfermedad de Alzheimer/genética , Animales , Células Cultivadas , Secuenciación de Inmunoprecipitación de Cromatina , Eucromatina/genética , Femenino , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Ontología de Genes , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Madre Pluripotentes/metabolismo , Complejo Represivo Polycomb 1/genética , Empalme del ARN/genética
12.
Nat Commun ; 12(1): 1859, 2021 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-33767140

RESUMEN

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoproteins initiates co-transcriptionally and requires the action of the assembly machinery including the Hsp90/R2TP complex, the Rsa1p:Hit1p heterodimer and the Bcd1 protein. We present genetic interactions between the Rsa1p-encoding gene and genes involved in chromatin organization including RTT106 that codes for the H3-H4 histone chaperone Rtt106p controlling H3K56ac deposition. We show that Bcd1p binds Rtt106p and controls its transcription-dependent recruitment by reducing its association with RNA polymerase II, modulating H3K56ac levels at gene body. We reveal the 3D structures of the free and Rtt106p-bound forms of Bcd1p using nuclear magnetic resonance and X-ray crystallography. The interaction is also studied by a combination of biophysical and proteomic techniques. Bcd1p interacts with a region that is distinct from the interaction interface between the histone chaperone and histone H3. Our results are evidence for a protein interaction interface for Rtt106p that controls its transcription-associated activity.


Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Chaperonas Moleculares/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Activación Transcripcional/fisiología , Proliferación Celular/fisiología , Cromatina/genética , Cristalografía por Rayos X , Histonas/metabolismo , Resonancia Magnética Nuclear Biomolecular , ARN Polimerasa II/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/genética , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transcripción Genética/genética
13.
Nat Commun ; 12(1): 1865, 2021 03 25.
Artículo en Inglés | MEDLINE | ID: mdl-33767158

RESUMEN

Pluripotent cells of the mammalian embryo undergo extensive chromatin rewiring to prepare for lineage commitment after implantation. Repressive H3K27me3, deposited by Polycomb Repressive Complex 2 (PRC2), is reallocated from large blankets in pre-implantation embryos to mark promoters of developmental genes. The regulation of this global redistribution of H3K27me3 is poorly understood. Here we report a post-translational mechanism that destabilizes PRC2 to constrict H3K27me3 during lineage commitment. Using an auxin-inducible degron system, we show that the deubiquitinase Usp9x is required for mouse embryonic stem (ES) cell self-renewal. Usp9x-high ES cells have high PRC2 levels and bear a chromatin and transcriptional signature of the pre-implantation embryo, whereas Usp9x-low ES cells resemble the post-implantation, gastrulating epiblast. We show that Usp9x interacts with, deubiquitinates and stabilizes PRC2. Deletion of Usp9x in post-implantation embryos results in the derepression of genes that normally gain H3K27me3 after gastrulation, followed by the appearance of morphological abnormalities at E9.5, pointing to a recurrent link between Usp9x and PRC2 during development. Usp9x is a marker of "stemness" and is mutated in various neurological disorders and cancers. Our results unveil a Usp9x-PRC2 regulatory axis that is critical at peri-implantation and may be redeployed in other stem cell fate transitions and disease states.


Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Células Madre Embrionarias de Ratones/citología , Células Madre Pluripotentes/citología , Complejo Represivo Polycomb 2/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Animales , Células Cultivadas , Cromatina/metabolismo , Femenino , Histonas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Interferencia de ARN , ARN Interferente Pequeño/genética , Ubiquitina Tiolesterasa/genética
14.
Nat Commun ; 12(1): 1956, 2021 03 29.
Artículo en Inglés | MEDLINE | ID: mdl-33782403

RESUMEN

Nucleophosmin (NPM1) is the most commonly mutated gene in acute myeloid leukemia (AML) resulting in aberrant cytoplasmic translocation of the encoded nucleolar protein (NPM1c+). NPM1c+ maintains a unique leukemic gene expression program, characterized by activation of HOXA/B clusters and MEIS1 oncogene to facilitate leukemogenesis. However, the mechanisms by which NPM1c+ controls such gene expression patterns to promote leukemogenesis remain largely unknown. Here, we show that the activation of HOXBLINC, a HOXB locus-associated long non-coding RNA (lncRNA), is a critical downstream mediator of NPM1c+-associated leukemic transcription program and leukemogenesis. HOXBLINC loss attenuates NPM1c+-driven leukemogenesis by rectifying the signature of NPM1c+ leukemic transcription programs. Furthermore, overexpression of HoxBlinc (HoxBlincTg) in mice enhances HSC self-renewal and expands myelopoiesis, leading to the development of AML-like disease, reminiscent of the phenotypes seen in the Npm1 mutant knock-in (Npm1c/+) mice. HoxBlincTg and Npm1c/+ HSPCs share significantly overlapped transcriptome and chromatin structure. Mechanistically, HoxBlinc binds to the promoter regions of NPM1c+ signature genes to control their activation in HoxBlincTg HSPCs, via MLL1 recruitment and promoter H3K4me3 modification. Our study reveals that HOXBLINC lncRNA activation plays an essential oncogenic role in NPM1c+ leukemia. HOXBLINC and its partner MLL1 are potential therapeutic targets for NPM1c+ AML.


Asunto(s)
Carcinogénesis/genética , Regulación Leucémica de la Expresión Génica , Proteínas de Homeodominio/genética , Leucemia Mieloide Aguda/genética , Proteínas Nucleares/genética , ARN Largo no Codificante/genética , Animales , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular , Perfilación de la Expresión Génica , Xenoinjertos , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/genética , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Ratones Transgénicos , Familia de Multigenes , Mutación , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/genética , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide/metabolismo , Proteína de la Leucemia Mieloide-Linfoide/genética , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Mielopoyesis/genética , Proteínas Nucleares/deficiencia , Regiones Promotoras Genéticas , ARN Largo no Codificante/agonistas , ARN Largo no Codificante/metabolismo , Transducción de Señal , Transcripción Genética
15.
Int J Mol Sci ; 22(4)2021 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-33669725

RESUMEN

Trichostatin A ([R-(E,E)]-7-[4-(dimethylamino) phenyl]-N-hydroxy- 4,6-dimethyl- 7-oxo-2,4-heptadienamide, TSA) affects chromatin state through its potent histone deacetylase inhibitory activity. Interfering with the removal of acetyl groups from lysine residues in histones is one of many epigenetic regulatory processes that control gene expression. Histone deacetylase inhibition drives cells toward the differentiation stage, favoring the activation of specific genes. In this paper, we investigated the effects of TSA on H3 and H4 lysine acetylome and methylome profiling in mice embryonic stem cells (ES14), treated with trichostatin A (TSA) by using a new, untargeted approach, consisting of trypsin-limited proteolysis experiments coupled with MALDI-MS and LC-MS/MS analyses. The method was firstly set up on standard chicken core histones to probe the optimized conditions in terms of enzyme:substrate (E:S) ratio and time of proteolysis and, then, applied to investigate the global variations of the acetylation and methylation state of lysine residues of H3 and H4 histone in the embryonic stem cells (ES14) stimulated by TSA and addressed to differentiation. The proposed strategy was found in its simplicity to be extremely effective in achieving the identification and relative quantification of some of the most significant epigenetic modifications, such as acetylation and lysine methylation. Therefore, we believe that it can be used with equal success in wider studies concerning the characterization of all epigenetic modifications.


Asunto(s)
Células Madre Embrionarias/metabolismo , Histonas/metabolismo , Ácidos Hidroxámicos/farmacología , Lisina/metabolismo , Acetilación/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Dimetilsulfóxido/farmacología , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Histonas/química , Metilación/efectos de los fármacos , Ratones , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Péptidos/química , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteolisis/efectos de los fármacos
16.
Int J Mol Sci ; 22(4)2021 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-33670779

RESUMEN

Histone deacetylases (HDACs) are conserved enzymes that remove acetyl groups from lysine side chains in histones and other proteins and play a crucial role in epigenetic regulation. Previously, we showed that histone acetylation is implicated in ultraviolet (UV)-induced inflammation and matrix impairment. To elucidate the histone acetylation status and specific HDACs involved in skin aging, we examined the changes in histone acetylation, global HDAC activity, and the expression of HDACs and sirtuins (SIRTs) in intrinsically aged and photoaged human skin as well as in UV-irradiated human skin in vivo. Following acute UV irradiation, the acetylated histone H3 (AcH3) level was increased, but HDAC activity and the expression levels of HDAC4, HDAC11, and SIRT4 were significantly decreased. In intrinsically aged skin, AcH3 levels were increased, but HDAC activity and the expression levels of HDAC4, HDAC5, HDAC10, HDAC11, SIRT6, and SIRT7 were significantly decreased. However, histone acetylation and HDAC expression in photoaged skin were not significantly different from those in intrinsically aged skin. Collectively, HDAC4 and HDAC11 were decreased in both UV-irradiated and intrinsically aged skin, suggesting that they may play a universal role in increased histone acetylation associated with skin aging.


Asunto(s)
Histona Desacetilasas/metabolismo , Histonas/metabolismo , Envejecimiento de la Piel/efectos de la radiación , Rayos Ultravioleta , Acetilación/efectos de la radiación , Humanos , Proteínas Mitocondriales/metabolismo , Sirtuinas/metabolismo , Piel/metabolismo , Piel/efectos de la radiación
17.
Int J Mol Sci ; 22(4)2021 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-33671470

RESUMEN

G-quadruplexes (G4s) are four-stranded helical structures that regulate several nuclear processes, including gene expression and telomere maintenance. We observed that G4s are located in GC-rich (euchromatin) regions and outside the fibrillarin-positive compartment of nucleoli. Genomic regions around G4s were preferentially H3K9 acetylated and H3K9 dimethylated, but H3K9me3 rarely decorated G4 structures. We additionally observed the variability in the number of G4s in selected human and mouse cell lines. We found the highest number of G4s in human embryonic stem cells. We observed the highest degree of colocalization between G4s and transcription factories, positive on the phosphorylated form of RNA polymerase II (RNAP II). Similarly, a high colocalization rate was between G4s and nuclear speckles, enriched in pre-mRNA splicing factor SC-35. PML bodies, the replication protein SMD1, and Cajal bodies colocalized with G4s to a lesser extent. Thus, G4 structures seem to appear mainly in nuclear compartments transcribed via RNAP II, and pre-mRNA is spliced via the SC-35 protein. However, α-amanitin, an inhibitor of RNAP II, did not affect colocalization between G4s and transcription factories as well as G4s and SC-35-positive domains. In addition, irradiation by γ-rays did not change a mutual link between G4s and DNA repair proteins (G4s/γH2AX, G4s/53BP1, and G4s/MDC1), accumulated into DNA damage foci. Described characteristics of G4s seem to be the manifestation of pronounced G4s stability that is likely maintained not only via a high-order organization of these structures but also by a specific histone signature, including H3K9me2, responsible for chromatin compaction.


Asunto(s)
Núcleo Celular/metabolismo , G-Cuádruplex , Histonas/metabolismo , Transcripción Genética , Acetilación , Animales , Composición de Base/genética , Línea Celular , Nucléolo Celular/metabolismo , Cromatina/metabolismo , ADN/metabolismo , Reparación del ADN , Epigénesis Genética , Humanos , Cuerpos de Inclusión/metabolismo , Metilación , Ratones
18.
Nat Commun ; 12(1): 1763, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741944

RESUMEN

Accurate chromosome segregation relies on the specific centromeric nucleosome-kinetochore interface. In budding yeast, the centromere CBF3 complex guides the deposition of CENP-A, an H3 variant, to form the centromeric nucleosome in a DNA sequence-dependent manner. Here, we determine the structures of the centromeric nucleosome containing the native CEN3 DNA and the CBF3core bound to the canonical nucleosome containing an engineered CEN3 DNA. The centromeric nucleosome core structure contains 115 base pair DNA including a CCG motif. The CBF3core specifically recognizes the nucleosomal CCG motif through the Gal4 domain while allosterically altering the DNA conformation. Cryo-EM, modeling, and mutational studies reveal that the CBF3core forms dynamic interactions with core histones H2B and CENP-A in the CEN3 nucleosome. Our results provide insights into the structure of the budding yeast centromeric nucleosome and the mechanism of its assembly, which have implications for analogous processes of human centromeric nucleosome formation.


Asunto(s)
Centrómero/metabolismo , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Cinetocoros/metabolismo , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Secuencia de Aminoácidos , Centrómero/genética , Centrómero/ultraestructura , Proteína A Centromérica/química , Proteína A Centromérica/genética , Proteína A Centromérica/metabolismo , Microscopía por Crioelectrón , ADN de Hongos/química , ADN de Hongos/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Cinetocoros/química , Conformación de Ácido Nucleico , Nucleosomas/genética , Nucleosomas/ultraestructura , Unión Proteica , Dominios Proteicos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido
19.
Nat Commun ; 12(1): 1965, 2021 03 30.
Artículo en Inglés | MEDLINE | ID: mdl-33785750

RESUMEN

Exploring spatial organization and relationship of diverse biomolecules within cellular nanoenvironments is important to elucidate the fundamental processes of life. However, it remains methodologically challenging. Herein, we report a molecular recognition mechanism cellular macromolecules-tethered DNA walking indexing (Cell-TALKING) to probe the nanoenvironments containing diverse chromatin modifications. As an example, we characterize the nanoenvironments of three DNA modifications around one histone posttranslational modification (PTM). These DNA modifications in fixed cells are labeled with respective DNA barcoding probes, and then the PTM site is tethered with a DNA walking probe. Cell-TALKING can continuously produce cleavage records of any barcoding probes nearby the walking probe. New 3'-OH ends are generated on the cleaved barcoding probes to induce DNA amplification for downstream detections. Combining fluorescence imaging, we identify various combinatorial chromatin modifications and investigate their dynamic changes during cell cycles. We also explore the nanoenvironments in different cancer cell lines and clinical specimens. In principle, using high-throughput sequencing instead of fluorescence imaging may allow the detection of complex cellular nanoenvironments containing tens of biomolecules such as transcription factors.


Asunto(s)
Microambiente Celular/genética , Cromatina/genética , ADN/genética , Epigénesis Genética , Cromatina/metabolismo , ADN/metabolismo , Técnicas Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Histonas/metabolismo , Humanos , Biología Molecular/métodos , Procesamiento Proteico-Postraduccional , Reproducibilidad de los Resultados , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
20.
Int J Mol Sci ; 22(4)2021 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-33670267

RESUMEN

The Helicase-related protein 3 (Hrp3), an ATP-dependent chromatin remodeling enzyme from the CHD family, is crucial for maintaining global nucleosome occupancy in Schizosaccharomyces pombe (S. pombe). Although the ATPase domain of Hrp3 is essential for chromatin remodeling, the contribution of non-ATPase domains of Hrp3 is still unclear. Here, we investigated the role of non-ATPase domains using in vitro methods. In our study, we expressed and purified recombinant S. pombe histone proteins, reconstituted them into histone octamers, and assembled nucleosome core particles. Using reconstituted nucleosomes and affinity-purified wild type and mutant Hrp3 from S. pombe we created a homogeneous in vitro system to evaluate the ATP hydrolyzing capacity of truncated Hrp3 proteins. We found that all non-ATPase domain deletions (∆chromo, ∆SANT, ∆SLIDE, and ∆coupling region) lead to reduced ATP hydrolyzing activities in vitro with DNA or nucleosome substrates. Only the coupling region deletion showed moderate stimulation of ATPase activity with the nucleosome. Interestingly, affinity-purified Hrp3 showed co-purification with all core histones suggesting a strong association with the nucleosomes in vivo. However, affinity-purified Hrp3 mutant with SANT and coupling regions deletion showed complete loss of interactions with the nucleosomes, while SLIDE and chromodomain deletions reduced Hrp3 interactions with the nucleosomes. Taken together, nucleosome association and ATPase stimulation by DNA or nucleosomes substrate suggest that the enzymatic activity of Hrp3 is fine-tuned by unique contributions of all four non-catalytic domains.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Nucleosomas/metabolismo , Schizosaccharomyces/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfato/química , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Histonas/química , Histonas/genética , Histonas/metabolismo , Nucleosomas/química , Nucleosomas/genética , Schizosaccharomyces/química , Schizosaccharomyces/genética , Eliminación de Secuencia
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