Gene expression is regulated at many levels. Most regulation, however, is believed to occur at the level of transcription initiation. Transcription factors, chromatin-modifying enzymes, and basal transcription factors unite to activate genes and are recruited in a precise order to promoters. This thesis focuses on mutations in the promoter region as a putative cause of disturbed transcriptional regulation. We report on the characterization of an important regulatory element (PKR-RE1) in the erythroid-specific promoter of the human hexokinase gene (HK1). In addition, we describe the role of exon 1 in erythroid-specific transcriptional regulation of the human gene for protoporphyrinogen oxidase (PPOX). As this exon is part of the 5’untranslated region, PPOX is an example of how tissue specific regulation of a gene can be achieved by a single promoter and tissue specific enhancers. Both studies are examples of basal promoter research, which serves as the basis of all knowledge regarding transcriptional regulation of a gene. This knowledge is essential to investigate if promoter mutations and sequence variations, identified in a molecular diagnostic search for disease causing mutations in patients, or in epidemiologic studies with fairly healthy subjects, are putatively disease causing. Following this, we describe a family with a number of gamma-globin promoter sequence variations and we discuss the putative association with Hereditary Persistence of Fetal Hemoglobin (HPFH). We used transient transfection assays to study one of the identified mutations, a substitution (-16C>G) present in cis in the promoters of both human gamma-globin genes. Transient transfection assays showed that it does not affect gamma-globin expression in the fetal nor in the adult stage of development. In addition, we functional characterized a common polymorphic variation in the erythroid-specific promoter of PKLR, because of a suspected association with low PK activity levels in a number of patients. Again we showed, by use of functional assays, that this polymorphic variation does not decrease promoter activity in vitro. Both studies therefore emphasize the importance of using functional assays to investigate polymorphic promoter variations. In addition, we investigated the –193A>G mutation in the erythroid-specific promoter of HK1, in a patient with hexokinase deficiency and chronic hemolysis. We showed that the –193A>G mutation strongly down-regulates HK1 promoter activity and that binding of AP1/c-jun to the promoter of HK1 is disrupted by this mutation. The effect of promoter mutations can be very subtle. In addition, promoter mutation analysis is complex and often laborious and difficult to perform. Therefore, thorough studies of promoter mutations are scarce and often confined to basic promoter research laboratories. Despite the complex nature of the concerning assays, promoter analysis is necessary to improve molecular diagnostics of disease-causing promoter mutations. In addition, promoter mutation analysis improves our understanding of the role of disturbed transcriptional regulation in human disease. Therefore, we believe that routine laboratories should team up with basal promoter research groups, to improve the molecular diagnostics of patients with disease-causing promoter mutations
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