RESUMEN
Myst family genes encode lysine acetyltransferases that mainly mediate histone acetylation to control transcription, DNA replication and DNA damage response. They form tetrameric complexes with PHD-finger proteins (Brpfs or Jades) and small non-catalytic subunits Ing4/5 and Meaf6. Although all the components of the complex are well-conserved from yeast to mammals, the function of Meaf6 and its homologs has not been elucidated in any species. Here we revealed the role of Meaf6 utilizing inducible Meaf6 KO ES cells. By elimination of Meaf6, proliferation ceased although histone acetylations were largely unaffected. In the absence of Meaf6, one of the Myst family members Myst2/Kat7 increased the ability to interact with PHD-finger proteins. This study is the first indication of the function of Meaf6, which shows it is not essential for HAT activity but modulates the assembly of the Kat7 complex.
Asunto(s)
Células Madre Embrionarias/metabolismo , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Acetilación , Alelos , Animales , Secuencia de Bases , Línea Celular , Proliferación Celular , Replicación del ADN , Células Madre Embrionarias/citología , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Ontología de Genes , Histona Acetiltransferasas/genética , Histonas/genética , Ratones , Anotación de Secuencia MolecularRESUMEN
BACKGROUND: Stable expression of transgenes is an important technique to analyze gene function. Various drug resistance genes, such as neo, pac, hph, zeo, bsd, and hisD, have been equally used as selection markers to isolate a transfectant without considering their dose-dependent characters. RESULTS: We quantitatively measured the variation of transgene expression levels in mouse embryonic stem (mES) cells, using a series of bi-cistronic expression vectors that contain Egfp expression cassette linked to each drug resistant gene via IRES with titration of the selective drugs, and found that the transgene expression levels achieved in each system with this vector design are in order, in which pac and zeo show sharp selection of transfectants with homogenously high expression levels. We also showed the importance of the choice of the drug selection system in gene-trap or gene targeting according to this order. CONCLUSIONS: The results of the present study clearly demonstrated that an appropriate choice of the drug resistance gene(s) is critical for a proper design of the experimental strategy.
Asunto(s)
Células Madre Embrionarias/fisiología , Marcación de Gen/métodos , Proteínas Recombinantes de Fusión/análisis , Transgenes , Animales , Biotecnología/métodos , Resistencia a Medicamentos/genética , Células Madre Embrionarias/metabolismo , Marcadores Genéticos/genética , Proteínas Fluorescentes Verdes/análisis , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Cinética , Ratones , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Selección GenéticaRESUMEN
Preparation of specific lineages at high purities from embryonic stem (ES) cells requires both selective culture conditions and markers to guide and monitor the differentiation. In this study, we distinguished definitive and visceral endoderm by using a mouse ES cell line that bears the gfp and human IL2R alpha (also known as CD25) marker genes in the goosecoid (Gsc) and Sox17 loci, respectively. This cell line allowed us to monitor the generation of Gsc+ Sox17+ definitive endoderm and Gsc- Sox17+ visceral endoderm and to define culture conditions that differentially induce definitive and visceral endoderm. By comparing the gene expression profiles of definitive and visceral endoderm, we identified seven surface molecules that are expressed differentially in the two populations. One of the seven markers, Cxcr4, to which a monoclonal antibody is available allowed us to monitor and purify the Gsc+ population from genetically unmanipulated ES cells under the condition that selects definitive endoderm.