RESUMO
Active DNA demethylation via ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming in cell state transitions. TET enzymes catalyze up to three successive oxidations of 5-methylcytosine (5mC), generating 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), or 5-carboxycytosine (5caC). Although these bases are known to contribute to distinct demethylation pathways, the lack of tools to uncouple these sequential oxidative events has constrained our mechanistic understanding of the role of TETs in chromatin reprogramming. Here, we describe the first application of biochemically engineered TET mutants that unlink 5mC oxidation steps, examining their effects on somatic cell reprogramming. We show that only TET enzymes proficient for oxidation to 5fC/5caC can rescue the reprogramming potential of Tet2-deficient mouse embryonic fibroblasts. This effect correlated with rapid DNA demethylation at reprogramming enhancers and increased chromatin accessibility later in reprogramming. These experiments demonstrate that DNA demethylation through 5fC/5caC has roles distinct from 5hmC in somatic reprogramming to pluripotency.
Assuntos
5-Metilcitosina/metabolismo , Reprogramação Celular , Proteínas de Ligação a DNA/metabolismo , Embrião de Mamíferos/metabolismo , Elementos Facilitadores Genéticos , Epigênese Genética , Fibroblastos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Dioxigenases , Embrião de Mamíferos/citologia , Fibroblastos/citologia , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Mutação , Células NIH 3T3 , Proteínas Proto-Oncogênicas/genéticaRESUMO
Transient receptor potential (TRP) cation channel genes code for an extensive family of conserved proteins responsible for a variety of physiological processes, including sensory perception, ion homeostasis, and chemical signal transduction. The TRP superfamily consists of seven subgroups, one of which is the transient receptor potential vanilloid (trpv) channel family. While trpv channels are relatively well studied in adult vertebrate organisms given their role in functions such as nociception, thermoregulation, and osmotic regulation in mature tissues and organ systems, relatively little is known regarding their function during embryonic development. Although there are some reports of the expression of specific trpv channels at particular stages in various organisms, there is currently no comprehensive analysis of trpv channels during embryogenesis. Here, performing in situ hybridization, we examined the spatiotemporal expression of trpv channel mRNA during early Xenopus laevis embryogenesis. Trpv channels exhibited unique patterns of embryonic expression at distinct locations including the trigeminal ganglia, spinal cord, cement gland, otic vesicle, optic vesicle, nasal placode, notochord, tailbud, proctodeum, branchial arches, epithelium, somite and the animal pole during early development. We have also observed the colocalization of trpv channels at the animal pole (trpv 2/4), trigeminal ganglia (trpv 1/2), and epithelium (trpv 5/6). These localization patterns suggest that trpv channels may play diverse roles during early embryonic development.
Assuntos
Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Canais de Potencial de Receptor Transitório/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Animais , Embrião não Mamífero/citologia , Desenvolvimento Embrionário , Canais de Potencial de Receptor Transitório/genética , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMO
We report the genome sequences of 14 cluster K mycobacteriophages isolated using Mycobacterium smegmatis mc²155 as host. Four are closely related to subcluster K1 phages, and 10 are members of subcluster K6. The phage genomes span considerable sequence diversity, including multiple types of integrases and integration sites.
