http://repub.eur.nl/res/aut/8173/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/13423/ 2004-06-15T00:00:00Z The human beta-globin locus control region (LCR) is required for the maintenance of an open chromatin configuration of the locus. It interacts with the genes and the hypersensitive regions flanking the locus to form an active chromatin hub (ACH) transcribing the genes. Proper developmental control of globin genes is largely determined by gene proximal regulatory sequences. Here, we provide the first functional evidence of the role of the most active sites of the LCR and the promoter of the beta-globin gene in the maintenance of the ACH. When the human beta-globin gene promoter is deleted in the context of a full LCR, the ACH is maintained with the beta-globin gene remaining in proximity. Additional deletion of hypersensitive site HS3 or HS2 of the LCR shows that HS3, but not HS2, in combination with the beta-globin promoter is crucial for the maintenance of the ACH at the definitive stage. We conclude that multiple interactions between the LCR and the beta-globin gene are required to maintain the appropriate spatial configuration in vivo. http://repub.eur.nl/res/pub/2640/ 2004-02-01T00:00:00Z OBJECTIVE: Persistent expression of the human fetal gamma-globin genes in the adult stage is often associated with naturally occurring deletions in the human beta-globin locus. The mapping of the 5' breakpoints of these deletions within the Agamma- to delta-globin intergenic region has suggested that regulatory elements involved in the silencing of the gamma-globin genes in the adult may be present. We previously identified two elements in this region, termed Enh and F, located 3' to the Agamma-globin gene acting as silencers in transient transfection assays. Here, we tested directly the in vivo significance of these elements in the developmental regulation of the human beta-like globin genes. MATERIALS AND METHODS. We selectively deleted both Enh and F elements in the context of a 185-kb human beta-globin locus PAC (P1 phage artificial chromosome) and tested the effects of this deletion on the expression of the human P-like globin genes in transgenic mice. RESULTS: The Enh/F deletion resulted in an increase in epsilon- and gamma-globin mRNA levels in the embryonic yolk sac stage of erythropoiesis, which appears to be due to an increase in the rate of transcription rather than to an increase in the number of cells transcribing the human globin locus. However, the human developmental switching from fetal gamma-globin to adult beta-globin gene expression in transgenic mice was not affected by this deletion. CONCLUSION: These results identify Enh and F as locus-wide regulatory elements capable of down-regulating transcription of the human beta-globin locus in an embryonic-specific manner. http://repub.eur.nl/res/pub/1146/ 2002-11-20T00:00:00Z Hemoglobin is the carrier of oxygen in the bloodstream. It is a tetrameric protein composed of two α-globin chains and two β-globin chains. These chains are encoded by the α- and β-globin gene loci. The globin loci are located on separate chromosomes and are composed of several developmentally regulated genes and regulatory elements located both upstream of the gene loci and around the individual genes. Lesions in the globin clusters give rise to blood disorders known as hemoglobinopathies. The study of the molecular basis of these disorders led to the identification of many regulatory elements of the globin loci, including the locus control region (LCR). The β-globin locus is one of the best-studied multi gene loci and has been extensively used as a model system for the study of gene regulation. The aims of this thesis are to study the mechanism of transcriptional activation of the globin loci, the role of the hypersensitive sites in LCR function and the further characterisation of putative γ-globin regulatory elements. In conclusion, the studies presented here show: 1. that there is evidence for a stochastic basis for activation of globin gene expression; 2. that the LCR does function as a holocomplex, but also that the individual hypersensitive sites act differently on each globin gene; 3. that the regulation of the individual globin genes is very complex and probably involves the interaction of various combinations of elements during different stages of development. http://repub.eur.nl/res/pub/9389/ 2000-01-01T00:00:00Z We report here modifications of human beta-globin PAC clones by homologous recombination in Escherichia coli DH10B, utilising a plasmid temperature sensitive for replication, the recA gene and a wild-type copy of the rpsL gene which allows for an efficient selection for plasmid loss in this host. High frequencies of recombination are observed even with very small lengths of homology and the method has general utility for introducing insertions, deletions and point mutations. No rearrangements were detected with the exception of one highly repetitive genomic sequence when either the E.COLI: RecA- or the lambdoid phage encoded RecT and RecE-dependent recombination systems were used.