RESUMEN
Covalent modifications of histone tails have fundamental roles in chromatin structure and function. Tri-methyl modification on lysine 27 of histone H3 (H3K27me3) usually correlates with gene repression that plays important roles in cell lineage commitment and development. Mash1 is a basic helix-loop-helix regulatory protein that plays a critical role in neurogenesis, where it expresses as an early marker. In this study, we have shown a decreased H3K27me3 accompanying with an increased demethylase of H3K27me3 (Jmjd3) at the promoter of Mash1 can elicit a dramatically efficient expression of Mash1 in RA-treated P19 cells. Over-expression of Jmjd3 in P19 cells also significantly enhances the RA-induced expression and promoter activity of Mash1. By contrast, the mRNA expression and promoter activity of Mash1 are significantly reduced, when Jmjd3 siRNA or dominant negative mutant of Jmjd3 is introduced into the P19 cells. Chromatin immunoprecipitation assays show that Jmjd3 is efficiently recruited to a proximal upstream region of Mash1 promoter that is overlapped with the specific binding site of Hes1 in RA-induced cells. Moreover, the association between Jmjd3 and Hes1 is shown in a co-Immunoprecipitation assay. It is thus likely that Jmjd3 is recruited to the Mash1 promoter via Hes1. Our results suggest that the demethylase activity of Jmjd3 and its mediator Hes1 for Mash1 promoter binding are both required for Jmjd3 enhanced efficient expression of Mash1 gene in the early stage of RA-induced neuronal differentiation of P19 cells.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/efectos de los fármacos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Tretinoina/farmacología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Línea Celular , Línea Celular Tumoral , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Immunoblotting , Inmunoprecipitación , Histona Demetilasas con Dominio de Jumonji/genética , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción HES-1RESUMEN
OBJECTIVE: To construct the eukaryotic expression plasmid of mouse histone lysine methyltransferase Setd7 and detect its effect on neuron development. METHODS: The clone of mouse Setd7 was obtained and inserted into the eukaryotic expression vector pCMV-3tag-6-Flag. The plasmid was transfected into HEK 293T and identified by Western blot. Real-time PCR was used to detect the effect of Setd7 on the neuron differentiation marker gene Ngn 1 mRNA expression. Dual luciferase reporter system was used to detect the effect of Setd7 on Ngn 1 mRNA expression. Real-time PCR was used to detect the effect of Setd 7 siRNA plasmid on Ngn 1 mRNA expression. RESULTS: An eukaryotic expression plasmid of Setd 7 was successfully constructed. Setd7 induced Ngn 1 mRNA expression and increased Ngn 1 promoter activity. Also, the knockdown of Setd 7 inhibited Ngn 1 mRNA expression. CONCLUSION: Histone lysine methyltransferase Setd7 can enhance neuron differentiation marker gene Ngn 1 transcription.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Proteínas del Tejido Nervioso/metabolismo , Proteína Metiltransferasas/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Regulación de la Expresión Génica , Vectores Genéticos , Células HEK293 , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Ratones , Proteínas del Tejido Nervioso/genética , Proteína Metiltransferasas/metabolismo , ARN Mensajero/genética , TransfecciónRESUMEN
The objective of this study was to determine the effect of exogenous mitochondria obtained from granulosa cells on the development of bovine embryos in vitro. We classified cumulus oocyte complexes (COCs) as good (G)- and poor (P)-quality oocytes based on cytoplasmic appearance and cumulus characteristics, and assessed mtDNA copy numbers in the G and P oocytes with real-time polymerase chain reaction (PCR). The mitochondria were isolated by fractionation and suspended in mitochondria injection buffer (MIB). Part one of the experiment consisted of the following treatments: (1) G-oocytes + sperm, (2) P-oocytes + mitochondria + MIB + sperm, (3) P-oocytes + MIB + sperm, and (4) P-oocytes + sperm. In part 2, oocytes were parthenogenetically activated. The treatments were: (1) G-oocytes, (2) P-oocytes + mitochondria + MIB, (3) P-oocytes + MIB, and (4) P-oocytes alone. The results indicated a significant difference in mtDNA copy number between G (361 113 +/- 147 114) and P (198 293 +/- 174 178) oocytes (p < 0.01). The rates of morula, blastocyst, and hatched blastocysts derived from P-oocytes + mitochondria were similar to those of G-oocytes, but significantly higher than P-oocytes without exogenous mitochondria in both the ICSI and parthenogenetic activation experiments. We found no difference in blastomere numbers between G-oocytes and P-oocytes + mitochondria in either experiment, but blastomere numbers in these two groups were significantly higher than in P-oocyte groups without exogenous mitochondria. These data suggest that mtDNA content is very important for early embryo development. Furthermore, the transfer of mitochondria from the same breed may improve embryo quality during preimplantation development.