RESUMEN
p53 is a tumour suppressor that regulates the cellular response to genotoxic stresses. p53 is a short-lived protein and its activity is regulated mostly by stabilization via different post-translational modifications. Here we report a novel mechanism of p53 regulation through lysine methylation by Set9 methyltransferase. Set9 specifically methylates p53 at one residue within the carboxyl-terminus regulatory region. Methylated p53 is restricted to the nucleus and the modification positively affects its stability. Set9 regulates the expression of p53 target genes in a manner dependent on the p53-methylation site. The crystal structure of a ternary complex of Set9 with a p53 peptide and the cofactor product S-adenosyl-l-homocysteine (AdoHcy) provides the molecular basis for recognition of p53 by this lysine methyltransferase.
Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , Lisina/metabolismo , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Aminoácidos , Apoptosis , Línea Celular , Núcleo Celular/metabolismo , Regulación de la Expresión Génica , Genes p53/genética , Genes ras/genética , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/química , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Metilación , Modelos Moleculares , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Unión Proteica , Conformación Proteica , Proteína Metiltransferasas , ARN Mensajero/genética , ARN Mensajero/metabolismo , S-Adenosilhomocisteína/metabolismo , Especificidad por Sustrato , TermodinámicaRESUMEN
Histone demethylase LSD1 (also known as KDM1 and AOF2) is active in various cancer cells, but its biological significance in human carcinogenesis is unexplored. In this study, we explored hypothesized interactions between LSD1 and MYPT1, a known regulator of RB1 phosphorylation. We found that MYPT1 was methylated in vitro and in vivo by histone lysine methyltransferase SETD7 and demethylated by LSD1, identifying Lys 442 of MYPT1 as a target for methylation/demethylation by these enzymes. LSD1 silencing increased MYPT1 protein levels, decreasing the steady state level of phosphorylated RB1 (Ser 807/811) and reducing E2F activity. MYPT1 methylation status influenced the affinity of MYPT1 for the ubiquitin-proteasome pathway of protein turnover. MYPT1 was unstable in murine cells deficient in SETD7, supporting the concept that MYPT1 protein stability is physiologically regulated by methylation status. LSD1 overexpression could activate RB1 phosphorylation by inducing a destabilization of MYPT1 protein. Taken together, our results comprise a novel cell cycle regulatory mechanism mediated by methylation/demethylation dynamics, and they reveal the significance of LSD1 overexpression in human carcinogenesis.
Asunto(s)
Histona Demetilasas/metabolismo , Quinasa de Cadena Ligera de Miosina/metabolismo , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Neoplasias/enzimología , Neoplasias/patología , Oxidorreductasas N-Desmetilantes/metabolismo , Animales , Ciclo Celular/fisiología , Humanos , Metilación , Ratones , Proteína de Retinoblastoma/metabolismoRESUMEN
Histone methylation and DNA methylation cooperatively regulate chromatin structure and gene activity. How these two systems coordinate with each other remains unclear. Here we study the biological function of lysine-specific demethylase 1 (LSD1, also known as KDM1 and AOF2), which has been shown to demethylate histone H3 on lysine 4 (H3K4) and lysine 9 (H3K9). We show that LSD1 is required for gastrulation during mouse embryogenesis. Notably, targeted deletion of the gene encoding LSD1 (namely, Aof2) in embryonic stem (ES) cells induces progressive loss of DNA methylation. This loss correlates with a decrease in DNA methyltransferase 1 (Dnmt1) protein, as a result of reduced Dnmt1 stability. Dnmt1 protein is methylated in vivo, and its methylation is enhanced in the absence of LSD1. Furthermore, Dnmt1 can be methylated by Set7/9 (also known as KMT7) and demethylated by LSD1 in vitro. Our findings suggest that LSD1 demethylates and stabilizes Dnmt1, thus providing a previously unknown mechanistic link between the histone and DNA methylation systems.
Asunto(s)
Metilación de ADN , Oxidorreductasas N-Desmetilantes/metabolismo , Animales , Diferenciación Celular , Proliferación Celular , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Desarrollo Embrionario , Células Madre Embrionarias/citología , Células Madre Embrionarias/enzimología , Histona Demetilasas , Ratones , Oxidorreductasas N-Desmetilantes/deficiencia , Especificidad por SustratoRESUMEN
The protein methyltransferase Set7/9 was recently shown to regulate p53 activity in cancer cells. However, the impact of Set7/9 on p53 function in vivo is unclear. To explore these issues, we created a null allele of Set7/9 in mice. Cells from Set7/9 mutant mice fail to methylate p53 K369, are unable to induce p53 downstream targets upon DNA damage, and are predisposed to oncogenic transformation. Importantly, we find that methylation of p53 by Set7/9 is required for the binding of the acetyltransferase Tip60 to p53 and for the subsequent acetylation of p53. We provide the first genetic evidence demonstrating that lysine methylation of p53 by Set7/9 is important for p53 activation in vivo and suggest a mechanistic link between methylation and acetylation of p53 through Tip60.