http://repub.eur.nl/res/aut/6223/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/8231/ 2003-01-01T00:00:00Z To further our understanding of the regulation of vertebrate globin loci, we have isolated cosmids containing alpha- and beta-globin genes from the pufferfish Fugu rubripes. By DNA fluorescence in situ hybridization (FISH) analysis, we show that Fugu contains 2 distinct hemoglobin loci situated on separate chromosomes. One locus contains only alpha-globin genes (alpha-locus), whereas the other also contains a beta-globin gene (alpha beta-locus). This is the first poikilothermic species analyzed in which the physical linkage of the alpha- and beta-globin genes has been uncoupled, supporting a model in which the separation of the alpha- and beta-globin loci has occurred through duplication of a locus containing both types of genes. Surveys for transcription factor binding sites and DNaseI hypersensitive site mapping of the Fugu alpha beta-locus suggest that a strong distal locus control region regulating the activity of the globin genes, as found in mammalian beta-globin clusters, may not be present in the Fugu alpha beta-locus. Searching the human and mouse genome databases with the genes surrounding the pufferfish hemoglobin loci reveals that homologues of some of these genes are proximal to cytoglobin, a recently described novel member of the globin family. This provides evidence that duplication of the globin loci has occurred several times during evolution, resulting in the 5 human globin loci known to date, each encoding proteins with specific functions in specific cell types. http://repub.eur.nl/res/pub/2587/ 2000-01-01T00:00:00Z Locus control regions (LCRs) alleviate chromatin-mediated transcriptional repression. Incomplete LCRs partially lose this property when integrated in transcriptionally restrictive genomic regions such as centromeres. This frequently results in position effect variegation (PEV), i.e. the suppression of expression in a proportion of the cells. Here we show that this PEV is influenced by the heterochromatic protein SUV39H1 and by the Polycomb group proteins M33 and BMI-1. A concentration variation of these proteins modulates the proportion of cells expressing human globins in a locus-dependent manner. Similarly, the transcription factors Sp1 or erythroid Krüppel-like factor (EKLF) also influence PEV, characterized by a change in the number of expressing cells and the chromatin structure of the locus. However, in contrast to results obtained in a euchromatic locus, EKLF influences the expression of the ?- more than the ?-globin genes, suggesting that the relief of silencing is caused by the binding of EKLF to the LCR and that genes at an LCR proximal position are more likely to be in an open chromatin state than genes at a distal position. http://repub.eur.nl/res/pub/12807/ 1999-02-15T00:00:00Z The analysis of a number of cases of beta-globin thalassemia and hereditary persistence of fetal hemoglobin (HPFH) due to large deletions in the beta-globin locus has led to the identification of several DNA elements that have been implicated in the switch from human fetal gamma- to adult beta-globin gene expression. We have tested this hypothesis for an element that covers the minimal distance between the thalassemia and HPFH deletions and is thought to be responsible for the difference between a deletion HPFH and deltabeta-thalassemia, located 5' of the delta-globin gene. This element has been deleted from a yeast artificial chromosome (YAC) containing the complete human beta-globin locus. Analysis of this modified YAC in transgenic mice shows that early embryonic expression is unaffected, but in the fetal liver it is subject to position effects. In addition, the efficiency of transcription of the beta-globin gene is decreased, but the developmental silencing of the gamma-globin genes is unaffected by the deletion. These results show that the deleted element is involved in the activation of the beta-globin gene perhaps through the loss of a structural function required for gene activation by long-range interactions.