RESUMO
Transcription factor control of cell-specific downstream targets can be significantly altered when the controlling factor is mutated. We show that the semi-dominant neonatal anemia (Nan) mutation in the EKLF/KLF1 transcription factor leads to ectopic expression of proteins that are not normally expressed in the red blood cell, leading to systemic effects that exacerbate the intrinsic anemia in the adult and alter correct development in the early embryo. Even when expressed as a heterozygote, the Nan-EKLF protein accomplishes this by direct binding and aberrant activation of genes encoding secreted factors that exert a negative effect on erythropoiesis and iron use. Our data form the basis for a novel mechanism of physiological deficiency that is relevant to human dyserythropoietic anemia and likely other disease states.
Assuntos
Anemia Neonatal/genética , Fatores de Transcrição Kruppel-Like/genética , Mutação , Substituição de Aminoácidos , Anemia Neonatal/sangue , Anemia Neonatal/embriologia , Animais , Animais Recém-Nascidos , Citocinas/sangue , DNA/genética , DNA/metabolismo , Modelos Animais de Doenças , Eritrócitos/metabolismo , Eritropoese/genética , Regulação da Expressão Gênica no Desenvolvimento , Heterozigoto , Humanos , Fatores de Transcrição Kruppel-Like/sangue , Fatores de Transcrição Kruppel-Like/deficiência , Camundongos , Camundongos Knockout , Camundongos Mutantes , Modelos Biológicos , Proteínas Musculares/sangue , Proteínas Mutantes/sangue , Proteínas Mutantes/genéticaRESUMO
Interstrand crosslinks (ICLs) are covalent lesions formed by cisplatin. The mechanism for the processing and removal of ICLs by DNA repair proteins involves nucleotide excision repair (NER), homologous recombination (HR) and fanconi anemia (FA) pathways. In this report, we monitored the processing of a flanking uracil adjacent to a cisplatin ICL by the proteins involved in the base excision repair (BER) pathway. Using a combination of extracts, purified proteins, inhibitors, functional assays and cell culture studies, we determined the specific BER proteins required for processing a DNA substrate with a uracil adjacent to a cisplatin ICL. Uracil DNA glycosylase (UNG) is the primary glycosylase responsible for the removal of uracils adjacent to cisplatin ICLs, whereas other uracil glycosylases can process uracils in the context of undamaged DNA. Repair of the uracil adjacent to cisplatin ICLs proceeds through the classical BER pathway, highlighting the importance of specific proteins in this redundant pathway. Removal of uracil is followed by the generation of an abasic site and subsequent cleavage by AP endonuclease 1 (APE1). Inhibition of either the repair or redox domain of APE1 gives rise to cisplatin resistance. Inhibition of the lyase domain of Polymerase ß (Polß) does not influence cisplatin cytotoxicity. In addition, lack of XRCC1 leads to increased DNA damage and results in increased cisplatin cytotoxicity. Our results indicate that BER activation at cisplatin ICLs influences crosslink repair and modulates cisplatin cytotoxicity via specific UNG, APE1 and Polß polymerase functions.
Assuntos
Cisplatino/toxicidade , Adutos de DNA , Reparo do DNA , Uracila-DNA Glicosidase/metabolismo , Uracila/metabolismo , Animais , DNA Polimerase beta/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Especificidade por Substrato , Proteína 1 Complementadora Cruzada de Reparo de Raio-XRESUMO
Understanding how transcriptional regulators are themselves controlled is important in attaining a complete picture of the intracellular effects that follow signaling cascades during early development and cell-restricted differentiation. We have addressed this issue by focusing on the regulation of EKLF/KLF1, a zinc finger transcription factor that plays a necessary role in the global regulation of erythroid gene expression. Using biochemical affinity purification, we have identified the DEK oncoprotein as a critical factor that interacts with an essential upstream enhancer element of the EKLF promoter and exerts a positive effect on EKLF levels. This element also binds a core set of erythroid transcription factors, suggesting that DEK is part of a tissue-restricted enhanceosome that contains BMP4-dependent and -independent components. Together with local enrichment of properly coded histones and an open chromatin domain, optimal transcriptional activation of the EKLF locus can be established.