RESUMEN
BACKGROUND: Apyrases are divalent ion dependent tri- and dinucleotide phosphatases with different substrate specificity. The intracellular lysosomal apyrase LALP70 is also expressed as a splice variant (LALP70v) lacking a VSFASSQQ motif in the center of the molecule (aminoacids 287-294). However, the functional significance of this motif is unknown. In this report we used a thin layer chromatography approach to study separately the UTPase and UDPase activity of the two LALP-enzymes. RESULTS: We show, that LALP70 and LALP70v cleaved UTP to UDP in a calcium independent manner. In contrast, the cleavage of UDP to UMP was strongly calcium dependent for LALP70, but calcium independent for LALP70v. CONCLUSIONS: The VSFASSQQ motif not only influences the substrate specificity of LALP70, but it confers calcium sensitivity to LALP70 during the UDP cleavage. Whether this is due to direct binding of calcium to this motif or to a conformational change of the enzyme, remains to be elucidated.
Asunto(s)
Apirasa/química , Calcio/farmacología , Pirofosfatasas/química , Secuencias de Aminoácidos , Animales , Apirasa/metabolismo , Células COS , Chlorocebus aethiops , Lisosomas/enzimología , Pirofosfatasas/metabolismo , Especificidad por Sustrato , Uridina Difosfato/metabolismo , Uridina Trifosfato/metabolismoRESUMEN
E2F6, a member of the E2F-family of transcription factors, is a retinoblastoma protein-independent transcriptional repressor. E2F6 associates with polycomb group (Pc-G) multiprotein complexes that contain histone H3 methyltransferases, suggesting that E2F6 represses genes by covalent histone modification. However, genes that are repressed by E2F6 via a mechanism that involves histone H3 methylation have not been identified. Using cDNA microarray experiments comparing wild-type and E2f6-/- mouse embryonic fibroblasts, we now found that E2F6 is required to silence the meiosis-specific genes SMC1beta and STAG3 in somatic cells. Re-expression of E2F6 in E2f6-/- cells was sufficient to restore their repression. E2F6 binds in vivo to the promoters of these genes through a conserved binding site. Transcriptional repression of SMC1beta and STAG3 by E2F6 involves multiple mechanisms, including methylation of histone H3 on lysine 9 and lysine 27. Our findings suggest a molecular mechanism for the stable transcriptional silencing of meiotic genes in somatic cells by E2F6.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Factor de Transcripción E2F6/genética , Silenciador del Gen , Proteínas Nucleares/genética , Acetilación , Animales , Secuencia de Bases , Células Cultivadas , Inmunoprecipitación de Cromatina , Metilación de ADN , ADN Complementario/metabolismo , Fibroblastos/metabolismo , Genes Reporteros , Histonas/química , Histonas/genética , Histonas/metabolismo , Immunoblotting , Lisina/química , Meiosis , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Oligonucleótidos/genética , Mutación Puntual , Regiones Promotoras Genéticas , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , TransgenesRESUMEN
E2F transcription factors play an important role in regulating mammalian cell proliferation. E2F6, the most recently identified E2F family member, is a transcriptional repressor. In an effort to ascertain the in vivo biological function of E2F6, we have generated an E2f6 mutant mouse strain. Mice lacking E2F6 are viable and healthy. Surprisingly, E2f6-/- embryonic fibroblasts proliferate normally. However, E2f6-/- animals display overt homeotic transformations of the axial skeleton that are strikingly similar to the skeletal transformations observed in polycomb mutant mice. This observation is compatible with the recent finding that endogenous E2F6 and one or more mammalian polycomb proteins are components of the same multiprotein complex. The accumulated evidence suggests that, during development, E2F6 participates in the recruitment of polycomb proteins to specific target promoters.