RESUMEN
Monomethylation of lysine 4 on histone H3 (H3K4me1) is a well-established feature of enhancers and promoters, although its function is unknown. Here, we uncover roles for H3K4me1 in diverse cell types. Remarkably, we find that MLL3/4 provokes monomethylation of promoter regions and the conditional repression of muscle and inflammatory response genes in myoblasts. During myogenesis, muscle genes are activated, lose MLL3 occupancy, and become H3K4-trimethylated through an alternative COMPASS complex. Monomethylation-mediated repression was not restricted to skeletal muscle. Together with H3K27me3 and H4K20me1, H3K4me1 was associated with transcriptional silencing in embryonic fibroblasts, macrophages, and human embryonic stem cells (ESCs). On promoters of active genes, we find that H3K4me1 spatially demarcates the recruitment of factors that interact with H3K4me3, including ING1, which, in turn, recruits Sin3A. Our findings point to a unique role for H3K4 monomethylation in establishing boundaries that restrict the recruitment of chromatin-modifying enzymes to defined regions within promoters.
Asunto(s)
Cromatina , Regulación del Desarrollo de la Expresión Génica , Histonas/genética , Desarrollo de Músculos/genética , Mioblastos/metabolismo , Animales , Metilación de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos , Fibroblastos/citología , Fibroblastos/metabolismo , Genoma , N-Metiltransferasa de Histona-Lisina , Histonas/metabolismo , Humanos , Proteína Inhibidora del Crecimiento 1 , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Mioblastos/citología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Transducción de Señal , Complejo Correpresor Histona Desacetilasa y Sin3 , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
We have examined changes in the chromatin landscape during muscle differentiation by mapping the genome-wide location of ten key histone marks and transcription factors in mouse myoblasts and terminally differentiated myotubes, providing an exceptionally rich dataset that has enabled discovery of key epigenetic changes underlying myogenesis. Using this compendium, we focused on a well-known repressive mark, histone H3 lysine 27 trimethylation, and identified novel regulatory elements flanking the myogenin gene that function as a key differentiation-dependent switch during myogenesis. Next, we examined the role of Polycomb-mediated H3K27 methylation in gene repression by systematically ablating components of both PRC1 and PRC2 complexes. Surprisingly, we found mechanistic differences between transient and permanent repression of muscle differentiation and lineage commitment genes and observed that the loss of PRC1 and PRC2 components produced opposing differentiation defects. These phenotypes illustrate striking differences as compared to embryonic stem cell differentiation and suggest that PRC1 and PRC2 do not operate sequentially in muscle cells. Our studies of PRC1 occupancy also suggested a "fail-safe" mechanism, whereby PRC1/Bmi1 concentrates at genes specifying nonmuscle lineages, helping to retain H3K27me3 in the face of declining Ezh2-mediated methyltransferase activity in differentiated cells.
Asunto(s)
Epigénesis Genética , Estudio de Asociación del Genoma Completo , Animales , Diferenciación Celular , Cromatina/metabolismo , Células Madre Embrionarias/citología , Histonas/metabolismo , Humanos , Lisina/química , Metilación , Ratones , Desarrollo de Músculos , Músculos/fisiología , Fenotipo , Proteínas del Grupo Polycomb , Proteínas Represoras/metabolismo , Transcripción GenéticaRESUMEN
We profile genome-wide histone 3 lysine 27 acetylation (H3K27ac) of 3 major brain cell types from hippocampus and dorsolateral prefrontal cortex (dlPFC) of subjects with and without Alzheimer's Disease (AD). We confirm that single nucleotide polymorphisms (SNPs) associated with late onset AD (LOAD) show a strong tendency to reside in microglia-specific gene regulatory elements. Despite this significant colocalization, we find that microglia harbor more acetylation changes associated with age than with amyloid-ß (Aß) load. In contrast, we detect that an oligodendrocyte-enriched glial (OEG) population contains the majority of differentially acetylated peaks associated with Aß load. These differential peaks reside near both early onset risk genes (APP, PSEN1, PSEN2) and late onset AD risk loci (including BIN1, PICALM, CLU, ADAM10, ADAMTS4, SORL1, FERMT2), Aß processing genes (BACE1), as well as genes involved in myelinating and oligodendrocyte development processes. Interestingly, a number of LOAD risk loci associated with differentially acetylated risk genes contain H3K27ac peaks that are specifically enriched in OEG. These findings implicate oligodendrocyte gene regulation as a potential mechanism by which early onset and late onset risk genes mediate their effects, and highlight the deregulation of myelinating processes in AD. More broadly, our dataset serves as a resource for the study of functional effects of genetic variants and cell type specific gene regulation in AD.
RESUMEN
DNA double-strand breaks (DSBs) are linked to neurodegeneration and senescence. However, it is not clear how DSB-bearing neurons influence neuroinflammation associated with neurodegeneration. Here, we characterize DSB-bearing neurons from the CK-p25 mouse model of neurodegeneration using single-nucleus, bulk, and spatial transcriptomic techniques. DSB-bearing neurons enter a late-stage DNA damage response marked by nuclear factor κB (NFκB)-activated senescent and antiviral immune pathways. In humans, Alzheimer's disease pathology is closely associated with immune activation in excitatory neurons. Spatial transcriptomics reveal that regions of CK-p25 brain tissue dense with DSB-bearing neurons harbor signatures of inflammatory microglia, which is ameliorated by NFκB knockdown in neurons. Inhibition of NFκB in DSB-bearing neurons also reduces microglia activation in organotypic mouse brain slice culture. In conclusion, DSBs activate immune pathways in neurons, which in turn adopt a senescence-associated secretory phenotype to elicit microglia activation. These findings highlight a previously unidentified role for neurons in the mechanism of disease-associated neuroinflammation.
Asunto(s)
Roturas del ADN de Doble Cadena , Microglía , Animales , Antivirales/metabolismo , ADN/metabolismo , Humanos , Ratones , Microglía/metabolismo , FN-kappa B/metabolismo , Neuronas/metabolismoRESUMEN
Neutralizing antibodies have become an important tool in treating infectious diseases. Recently, two separate approaches yielded successful antibody treatments for Ebola-one from genetically humanized mice and the other from a human survivor. Here, we describe parallel efforts using both humanized mice and convalescent patients to generate antibodies against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, which yielded a large collection of fully human antibodies that were characterized for binding, neutralization, and three-dimensional structure. On the basis of these criteria, we selected pairs of highly potent individual antibodies that simultaneously bind the receptor binding domain of the spike protein, thereby providing ideal partners for a therapeutic antibody cocktail that aims to decrease the potential for virus escape mutants that might arise in response to selective pressure from a single-antibody treatment.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Betacoronavirus/inmunología , Infecciones por Coronavirus/inmunología , Neumonía Viral/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Adolescente , Adulto , Enzima Convertidora de Angiotensina 2 , Animales , Anticuerpos Neutralizantes/química , Anticuerpos Antivirales/química , Afinidad de Anticuerpos , Citotoxicidad Celular Dependiente de Anticuerpos , Betacoronavirus/química , Sitios de Unión de Anticuerpos , Anticuerpos ampliamente neutralizantes/química , Anticuerpos ampliamente neutralizantes/inmunología , COVID-19 , Línea Celular , Infecciones por Coronavirus/terapia , Citofagocitosis , Epítopos , Humanos , Inmunización Pasiva , Ratones , Persona de Mediana Edad , Modelos Moleculares , Pruebas de Neutralización , Pandemias , Peptidil-Dipeptidasa A/metabolismo , Dominios y Motivos de Interacción de Proteínas , Receptores de Coronavirus , Receptores Virales/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Adulto Joven , Sueroterapia para COVID-19RESUMEN
The apolipoprotein E4 (APOE4) variant is the single greatest genetic risk factor for sporadic Alzheimer's disease (sAD). However, the cell-type-specific functions of APOE4 in relation to AD pathology remain understudied. Here, we utilize CRISPR/Cas9 and induced pluripotent stem cells (iPSCs) to examine APOE4 effects on human brain cell types. Transcriptional profiling identified hundreds of differentially expressed genes in each cell type, with the most affected involving synaptic function (neurons), lipid metabolism (astrocytes), and immune response (microglia-like cells). APOE4 neurons exhibited increased synapse number and elevated Aß42 secretion relative to isogenic APOE3 cells while APOE4 astrocytes displayed impaired Aß uptake and cholesterol accumulation. Notably, APOE4 microglia-like cells exhibited altered morphologies, which correlated with reduced Aß phagocytosis. Consistently, converting APOE4 to APOE3 in brain cell types from sAD iPSCs was sufficient to attenuate multiple AD-related pathologies. Our study establishes a reference for human cell-type-specific changes associated with the APOE4 variant. VIDEO ABSTRACT.
Asunto(s)
Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/metabolismo , Apolipoproteína E4/genética , Células Madre Pluripotentes Inducidas/metabolismo , Neuroglía/metabolismo , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/metabolismo , Apolipoproteína E3/metabolismo , Apolipoproteína E4/metabolismo , Astrocitos/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Sistemas CRISPR-Cas , Diferenciación Celular , Humanos , Metabolismo de los Lípidos , Microglía/inmunología , Microglía/metabolismo , Organoides/metabolismo , Fosfoproteínas/metabolismo , Transmisión Sináptica , TranscriptomaRESUMEN
The histone deacetylase HDAC2, which negatively regulates synaptic gene expression and neuronal plasticity, is upregulated in Alzheimer's disease (AD) patients and mouse models. Therapeutics targeting HDAC2 hold promise for ameliorating AD-related cognitive impairment; however, attempts to generate HDAC2-specific inhibitors have failed. Here, we take an integrative genomics approach to identify proteins that mediate HDAC2 recruitment to synaptic plasticity genes. Functional screening revealed that knockdown of the transcription factor Sp3 phenocopied HDAC2 knockdown and that Sp3 facilitated recruitment of HDAC2 to synaptic genes. Importantly, like HDAC2, Sp3 expression was elevated in AD patients and mouse models, where Sp3 knockdown ameliorated synaptic dysfunction. Furthermore, exogenous expression of an HDAC2 fragment containing the Sp3-binding domain restored synaptic plasticity and memory in a mouse model with severe neurodegeneration. Our findings indicate that targeting the HDAC2-Sp3 complex could enhance cognitive function without affecting HDAC2 function in other processes.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Histona Desacetilasa 2/metabolismo , Plasticidad Neuronal , Neuronas/metabolismo , Factor de Transcripción Sp3/metabolismo , Animales , Epigénesis Genética , Femenino , Código de Histonas , Histonas/genética , Histonas/metabolismo , Masculino , Memoria , Ratones , Neuronas/fisiología , Factor de Transcripción Sp3/genéticaRESUMEN
Mutations in MECP2 cause the neurodevelopmental disorder Rett syndrome (RTT). The RTT missense MECP2R306C mutation prevents MeCP2 from interacting with the NCoR/histone deacetylase 3 (HDAC3) complex; however, the neuronal function of HDAC3 is incompletely understood. We found that neuronal deletion of Hdac3 in mice elicited abnormal locomotor coordination, sociability and cognition. Transcriptional and chromatin profiling revealed that HDAC3 positively regulated a subset of genes and was recruited to active gene promoters via MeCP2. HDAC3-associated promoters were enriched for the FOXO transcription factors, and FOXO acetylation was elevated in Hdac3 knockout (KO) and Mecp2 KO neurons. Human RTT-patient-derived MECP2R306C neural progenitor cells had deficits in HDAC3 and FOXO recruitment and gene expression. Gene editing of MECP2R306C cells to generate isogenic controls rescued HDAC3-FOXO-mediated impairments in gene expression. Our data suggest that HDAC3 interaction with MeCP2 positively regulates a subset of neuronal genes through FOXO deacetylation, and disruption of HDAC3 contributes to cognitive and social impairment.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Histona Desacetilasas/genética , Proteína 2 de Unión a Metil-CpG/genética , Mutación/genética , Conducta Social , Animales , Humanos , Ratones Transgénicos , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Fenotipo , Síndrome de Rett/genéticaRESUMEN
The highly related mammalian Sin3A and Sin3B proteins provide a versatile platform for chromatin-modifying activities. Sin3-containing complexes play a role in gene repression through deacetylation of nucleosomes. Here, we explore a role for Sin3 in myogenesis by examining the phenotypes resulting from acute somatic deletion of both isoforms in vivo and from primary myotubes in vitro. Myotubes ablated for Sin3A alone, but not Sin3B, displayed gross defects in sarcomere structure that were considerably enhanced upon simultaneous ablation of both isoforms. Massively parallel sequencing of Sin3A- and Sin3B-bound genomic loci revealed a subset of target genes directly involved in sarcomere function that are positively regulated by Sin3A and Sin3B proteins. Both proteins were coordinately recruited to a substantial number of genes. Interestingly, depletion of Sin3B led to compensatory increases in Sin3A recruitment at certain target loci, but Sin3B was never found to compensate for Sin3A loss. Thus, our analyses describe a novel transcriptional role for Sin3A and Sin3B proteins associated with maintenance of differentiated muscle cells.
Asunto(s)
Desarrollo de Músculos/fisiología , Músculo Esquelético , Isoformas de Proteínas/metabolismo , Proteínas Represoras/metabolismo , Sarcómeros/fisiología , Animales , Línea Celular , Eliminación de Gen , Ratones , Músculo Esquelético/citología , Músculo Esquelético/fisiología , Fenotipo , Isoformas de Proteínas/genética , Interferencia de ARN , Proteínas Represoras/genética , Sarcómeros/ultraestructura , Complejo Correpresor Histona Desacetilasa y Sin3 , Tasa de SupervivenciaRESUMEN
Cytochromes P-450 2C11 and 2C13 are the major CYPs in rat liver microsomes. Despite a high degree of sequence identity, these two isozymes display different positional and regio-specific metabolism of steroid hormones, such as testosterone. CYP2C11 converts testosterone to 2alpha-hydroxyl and 16alpha-hydroxyl metabolites, while CYP2C13 produces primarily the 6beta-hydroxyl metabolite. Using a human CYP2C9 crystal structure as the template, homology models were generated for CYP2C11 and CYP2C13. Despite similar volume of the binding pockets for CYP2C11 and CYP2C13, the models for these two CYPs showed a substantial difference in the shape of the substrate-binding sites. Substrate docking using rigid and induced-fit methods showed that testosterone fits into the substrate-binding sites of both CYP2C11 and CYP2C13 without the need of added constraints. These docking exercises appear to support testosterone binding in both CYP2C11 and CYP2C13. A constrained docking using energy minimization is required to position testosterone for more precise positional and regio-specificity in supporting the observed metabolism. These results demonstrate the complexity of using modeling for understanding the binding of substrate to CYPs, and suggest that, as a complement to the metabolism data, modeling and docking may yield reliable structural information for the molecular interaction between the substrate and the CYPs.