RESUMEN
Chromatin accessibility is a hallmark of active regulatory regions and is functionally linked to transcriptional networks and cell identity. However, the molecular mechanisms and networks that govern chromatin accessibility have not been thoroughly studied. Here we conducted a genome-wide CRISPR screening combined with an optimized ATAC-see protocol to identify genes that modulate global chromatin accessibility. In addition to known chromatin regulators like CREBBP and EP400, we discovered a number of previously unrecognized proteins that modulate chromatin accessibility, including TFDP1, HNRNPU, EIF3D and THAP11 belonging to diverse biological pathways. ATAC-seq analysis upon their knockouts revealed their distinct and specific effects on chromatin accessibility. Remarkably, we found that TFDP1, a transcription factor, modulates global chromatin accessibility through transcriptional regulation of canonical histones. In addition, our findings highlight the manipulation of chromatin accessibility as an approach to enhance various cell engineering applications, including genome editing and induced pluripotent stem cell reprogramming.
Asunto(s)
Cromatina , Secuenciación de Nucleótidos de Alto Rendimiento , Cromatina/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Histonas/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Redes Reguladoras de GenesRESUMEN
Transgenic techniques have greatly increased our understanding of the transcriptional regulation of target genes through live reporter imaging, as well as the spatiotemporal function of a gene using loss- and gain-of-function constructs. In Xenopus species, two well-established transgenic methods, restriction enzyme-mediated integration and I-SceI meganuclease-mediated transgenesis, have been used to generate transgenic animals. However, donor plasmids are randomly integrated into the Xenopus genome in both methods. Here, we established a new and simple targeted transgenesis technique based on CRISPR/Cas9 in Xenopus laevis. In this method, Cas9 ribonucleoprotein (RNP) targeting a putative harbor site (the transforming growth factor beta receptor 2-like (tgfbr2l) locus) and a preset donor plasmid DNA were co-injected into the one-cell stage embryos of X. laevis. Approximately 10% of faithful reporter expression was detected in F0 crispants in a promoter/enhancer-specific manner. Importantly, efficient germline transmission and stable transgene expression were observed in the F1 offspring. The simplicity of this method only required preparation of a donor vector containing the tgfbr2l genome fragment and Cas9 RNP targeting this site, which are common experimental procedures used in Xenopus laboratories. Our improved technique allows the simple generation of transgenic X. laevis, so is expected to become a powerful tool for reporter assay and gene function analysis.
Asunto(s)
Sistemas CRISPR-Cas , Técnicas de Transferencia de Gen , Animales , Animales Modificados Genéticamente , Sistemas CRISPR-Cas/genética , Ribonucleoproteínas/genética , Transgenes , Xenopus laevis/genéticaRESUMEN
The promyelocytic leukemia (PML) body is a phase-separated nuclear structure physically associated with chromatin, implying its crucial roles in genome functions. However, its role in transcriptional regulation is largely unknown. We developed APEX-mediated chromatin labeling and purification (ALaP) to identify the genomic regions proximal to PML bodies. We found that PML bodies associate with active regulatory regions across the genome and with â¼300 kb of the short arm of the Y chromosome (YS300) in mouse embryonic stem cells. The PML body association with YS300 is essential for the transcriptional activity of the neighboring Y-linked clustered genes. Mechanistically, PML bodies provide specific nuclear spaces that the de novo DNA methyltransferase DNMT3A cannot access, resulting in the steady maintenance of a hypo-methylated state at Y-linked gene promoters. Our study underscores a new mechanism for gene regulation in the 3D nuclear space and provides insights into the functional properties of nuclear structures for genome function.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/metabolismo , Regulación de la Expresión Génica , Cuerpos de Inclusión Intranucleares/genética , Cromosoma Y/genética , Animales , Línea Celular , Cromatina/genética , Cromatina/metabolismo , ARN Helicasas DEAD-box/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN , ADN Metiltransferasa 3A , ADN-(Sitio Apurínico o Apirimidínico) Liasa/genética , Células Madre Embrionarias/fisiología , Endonucleasas/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Cuerpos de Inclusión Intranucleares/metabolismo , Ratones Noqueados , Antígenos de Histocompatibilidad Menor/genética , Enzimas Multifuncionales/genética , Familia de Multigenes , Estrés Oxidativo , Proteína de la Leucemia Promielocítica/genética , Proteína de la Leucemia Promielocítica/metabolismo , Proteínas/genética , Factores de Transcripción/genética , Cromosoma Y/metabolismoRESUMEN
Fyn-deficient pups born of Fyn-deficient parents die because they fail to suckle within 1-2 days after birth. Here we demonstrate that the neonatal death phenotype was influenced by the genetic background and an environmental odor. The odor of hexanal (C6-aldehyde) partially impaired mouse maternal behavior and induced the neonatal death of Fyn-deficient pups born of Fyn-deficient parents. This death phenotype was first observed in the breeding environment using autoclaved chips of Douglas fir. An analysis of the volatile chemicals in the autoclaved chips revealed an approximately 10-fold greater amount of hexanal than in non-autoclaved chips. Hexanal influenced the length of time virgin female mice engage in the maternal crouching behavior. In addition, Fyn-deficient females exhibited defects in the maternal behavior of nest building and pup retrieval, regardless of exposure to hexanal. These observations provide new insights into the regulation of maternal behavior by environmental and genetic factors.
