RESUMO
DNA methylation is an epigenetic modification that plays a vital role in a variety of biological processes, including the regulation of gene expression, cell differentiation, early embryonic development, genomic imprinting, and X chromosome inactivation. PGC7 is a maternal factor that maintains DNA methylation during early embryonic development. One mechanism of action has been identified by analyzing the interactions between PGC7 and UHRF1, H3K9 me2, or TET2/TET3, which reveals how PGC7 regulates DNA methylation in oocytes or fertilized embryos. However, the mechanism by which PGC7 regulates the post-translational modification of methylation-related enzymes remains to be elucidated. This study focused on F9 cells (embryonic cancer cells), which display high levels of PGC7 expression. We found that both knockdown of Pgc7 and inhibition of ERK activity resulted in increased genome-wide DNA methylation levels. Mechanistic experiments confirmed that inhibition of ERK activity led to the accumulation of DNMT1 in the nucleus, ERK phosphorylated DNMT1 at ser717, and DNMT1 Ser717-Ala mutation promoted the nuclear localization of DNMT1. Moreover, knockdown of Pgc7 also caused downregulation of ERK phosphorylation and promoted the accumulation of DNMT1 in the nucleus. In conclusion, we reveal a new mechanism by which PGC7 regulates genome-wide DNA methylation via phosphorylation of DNMT1 at ser717 by ERK. These findings may provide new insights into treatments for DNA methylation-related diseases.
Assuntos
Metilação de DNA , Epigênese Genética , Núcleo Celular/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , Impressão Genômica , Processamento de Proteína Pós-Traducional , Proteínas Cromossômicas não HistonaRESUMO
The gene dosage at the imprinted Dlk1-Dio3 locus is critical for cell growth and development. A relatively high gene expression within the Dlk1-Dio3 region, especially the active expression of Gtl2, has been identified as the only reliable marker for cell pluripotency. The DNA methylation state of the IG-DNA methylated regions (DMR), which is located upstream of the Gtl2 gene, dominantly contributes to the control of gene expression in the Dlk1-Dio3 locus. However, the precise mechanism underlying the regulation of DNA methylation in the IG-DMR remains largely unknown. Here, we use the F9 embryonal carcinoma cell line, a low pluripotent cell model, to identify the mechanism responsible for DNA methylation in the IG-DMR, and find that the interaction of PGC7 with UHRF1 is involved in maintaining DNA methylation and inducing DNA hypermethylation in the IG-DMR region. PGC7 and UHRF1 cooperatively bind in the IG-DMR to regulate the methylation of DNA and histones in this imprinted region. PGC7 promotes the recruitment of DNMT1 by UHRF1 to maintain DNA methylation in the IG-DMR locus. The interaction between PGC7 and UHRF1 strengthens their binding to H3K9me3 and leads to further enrichment of H3K9me3 in the IG-DMR by recruiting the specific histone methyltransferase SETDB1. Consequently, the abundance of H3K9me3 promotes DNMT3A to bind to the IG-DMR and increases DNA methylation level in this region. In summary, we propose a new mechanism of DNA methylation regulation in the IG-DMR locus and provide further insight into the understanding of the difference in Gtl2 expression levels between high and low pluripotent cells.
Assuntos
Metilação de DNA , RNA Longo não Codificante , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , DNA/metabolismo , Impressão Genômica , Histona Metiltransferases/genética , Histona Metiltransferases/metabolismo , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
FOXC1, a transcription factor involved in cell differentiation and embryogenesis, is demonstrated to be a negative regulator of Nanog in this study. FOXC1 is up-regulated in retinoic acid-induced differentiation of F9 Embryonal Carcinoma (EC) cells; furthermore, FOXC1 specifically inhibits the core pluripotency factor Nanog by binding to the proximal promoter. Overexpression of FOXC1 in F9 or knockdown in 3T3 results in the down-regulation or up-regulation of Nanog mRNA and proteins, respectively. In order to explain the mechanism by which FOXC1 inhibits Nanog expression, we identified the co-repressor HDAC2 from the FOXC1 interactome. FOXC1 recruits HDAC2 to Nanog promoter to decrease H3K27ac enrichment, resulting in transcription inhibition of Nanog. To the best of our knowledge, this is the first report that FOXC1 is involved in the epigenetic regulation of gene expression.
Assuntos
Células-Tronco de Carcinoma Embrionário/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona Desacetilase 2/metabolismo , Proteína Homeobox Nanog/genética , Regiões Promotoras Genéticas , Tretinoína/farmacologia , Animais , Antineoplásicos/farmacologia , Células-Tronco de Carcinoma Embrionário/efeitos dos fármacos , Células-Tronco de Carcinoma Embrionário/patologia , Epigênese Genética , Fatores de Transcrição Forkhead/genética , Células HEK293 , Histona Desacetilase 2/genética , Humanos , Camundongos , Células NIH 3T3 , Proteína Homeobox Nanog/metabolismoRESUMO
Intracellular pathogen resistance 1 (Ipr1) has been found to be a mediator to integrate cyclic GMP-AMP synthase (cGAS)-interferon regulatory factor 3 (IRF3), activated by intracellular pathogens, with the p53 pathway. Previous studies have shown the process of Ipr1 induction by various immune reactions, including intracellular bacterial and viral infections. The present study demonstrated that Ipr1 is regulated by the cGAS-IRF3 pathway during pathogenic infection. IRF3 was found to regulate Ipr1 expression by directly binding the interferon-stimulated response element motif of the Ipr1 promoter. Knockdown of Ipr1 decreased the expression of immunity-related GTPase family M member 1 (Irgm1), which plays critical roles in autophagy initiation. Irgm1 promoter characterization revealed a p53 motif in front of the transcription start site. P53 was found to participate in regulation of Irgm1 expression and IPR1-related effects on P53 stability by affecting interactions between ribosomal protein L11 (RPL11) and transformed mouse 3T3 cell double minute 2 (MDM2). Our results indicate that Ipr1 integrates cGAS-IRF3 with p53-modulated Irgm1 expression.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Nucleotídeos Cíclicos/metabolismo , Transativadores/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Citoplasma/metabolismo , Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/genética , Macrófagos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Células NIH 3T3 , Nucleotídeos Cíclicos/genética , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Células RAW 264.7 , Proteínas Ribossômicas/metabolismo , Transdução de Sinais , Transativadores/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
Hesx1, a homeobox gene expressed in embryonic stem cells (ESCs), has been implicated in the core transcription factors governing the pluripotent state. However, data about the underlying mechanism of how Hesx1 is involved in maintaining pluripotency is still scarce. In this study, we find Hesx1 responds to multiple pluripotency-related pathway inhibitors as well as LIF stimulation. Particularly, the expression of Hesx1 can be readily induced by dual inhibition (2i) of glycogen synthase kinase 3 and mitogen-activated protein kinase. Forced expression of Hesx1 can partially compensate for the withdrawal of either LIF or each component of 2i. We also demonstrate that LIF and each inhibitor of 2i can induce Hesx1 independent of one another. We tentatively put forward that Hesx1 is a common downstream target of LIF- and 2i-mediated self-renewal signaling pathways and plays an important role in maintaining ESC identity. Our study extends the methods of identifying the missing crucial factors in establishing ESC pluripotency.
