http://repub.eur.nl/res/aut/2916/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/2534/ 1997-01-01T00:00:00Z We have used gene competition to distinguish between possible mechanisms of transcriptional activation of the genes of the human beta-globin locus. The insertion of a second beta-globin gene at different points in the locus shows that the more proximal beta gene competes more effectively for activation by the locus control region (LCR). Reducing the relative distance between the genes and the LCR reduces the competitive advantage of the proximal gene, a result that supports activation by direct interaction between the LCR and the genes. Visualization of the primary transcripts shows that the level of transcription is proportional to the frequency of transcriptional periods and that such periods last approximately 8 min in vivo. We also find that the position of the beta-globin gene in the locus is important for correct developmental regulation. http://repub.eur.nl/res/pub/2516/ 1996-01-01T00:00:00Z Hereditary persistence of fetal haemoglobin (HPFH) is a clinically important condition in which a change in the developmental specificity of the gamma-globin genes results in varying levels of expression of fetal haemoglobin in the adult. The condition is benign and can significantly alleviate the symptoms of thalassaemia or sickle cell anaemia when co-inherited with these disorders. We have examined structure-function relationships in the -117 HPFH gamma promoter by analysing the effect of mutating specific promoter elements on the functioning of the wild-type and HPFH promoters. We find that CCAAT box mutants dramatically affect expression from the HPFH promoter in adult blood but have little effect on embryonic/fetal expression from the wild-type promoter. Our results suggest that there are substantial differences in the structure of the wild-type gamma promoter expressed early in development and the adult HPFH promoter. Together with previous results, this suggests that gamma silencing is a complex multifactorial phenomenon rather than being the result of a simple repressor binding to the promoter. We present a model for gamma-globin gene silencing that has significant implications for attempts to reactivate the gamma promoters in human adults by pharmacological means. http://repub.eur.nl/res/pub/2485/ 1993-04-01T00:00:00Z Recent studies indicate that different levels of control operate within multigene loci. In addition to regulatory sequences immediately flanking the genes, there are also elements that act over long distances on more than one gene. Competition for these elements among genes can influence both the level and timing of gene expression during development. http://repub.eur.nl/res/pub/2483/ 1993-01-01T00:00:00Z Transgenes linked to the beta-globin locus control region (LCR) are transcribed in a copy-dependent manner that is independent of the integration site. It has previously been shown that the LCR 5'HS2 region does not require its NF-E2 dimer binding site for LCR activity. In this paper we analyse synthetic 5'HS2 core constructs containing point mutations in the other factor binding sites 3' of the NF-E2 dimer site. The results show that 5'HS2 core is a partially active LCR that functions in a concatamer of at least two copies but not when present as a single copy in transgenic mice and that no single binding site within 5'HS2 is required for position-independent expression. In addition, the H-BP factor is identical to upstream stimulatory factor (USF) and full enhancement levels by 5'HS2 core in MEL cells require a combination of all the factor binding sites. We suggest that 5'HS2 cores in a concatamer interact with each other to establish an area of open chromatin and that this process may be the basis of LCR function. http://repub.eur.nl/res/pub/2476/ 1992-01-01T00:00:00Z In normal humans the fetal stage-specific gamma-globin genes are silenced after birth and not expressed in the adult. Exceptions are seen in cases of hereditary persistence of fetal haemoglobin (HPFH). These are clinically important because the elevated levels of gamma-globin can alleviate beta-thalassaemia and sickle cell anaemia. One class of mutations is associated with point mutations in the promoter of the gamma-globin genes (non-deletion HPFH), whereas others seem to be caused by large deletions 3' to the gamma-globin genes. To test whether the point mutation found in the Greek non-deletion HPFH (guanine to adenine at nucleotide position -117) is the cause of the raised gamma-globin levels in the adult stage and is not just a linked polymorphism, we engineered this mutation into a gamma-globin gene. When this gene was introduced into mice, the presence of the -117 mutation results in persistence of gamma-globin expression at a high level and a concomitant decrease in beta-globin expression in fetal and adult mice. We show that these changes correlate with the loss of binding of the transcription factor GATA1 to the gamma-globin promoter, suggesting that it may act as a negative regulator of the gamma-globin gene in adults. http://repub.eur.nl/res/pub/2478/ 1992-01-01T00:00:00Z We have used a linker-based ligation strategy to combine two 35-kb cosmid inserts from the human beta-globin locus into one linear fragment containing the entire locus. This 70-kb fragment was introduced into transgenic mice by microinjection of fertilized eggs. Southern blot analysis showed that a single complete transgene locus can be introduced into the germ line with high efficiency. Analysis of the expression patterns of the locus during development shows that the epsilon-globin gene behaves as a purely embryonic gene, the gamma-globin gene as an embryonic and early fetal gene, and the beta-globin gene as a fetal adult gene. Quantitation of expression showed that the levels of transcription of the epsilon- and gamma-globin genes are reversed relative to their mouse homologs but that the total output of the human and mouse loci is constant during development. These results suggest that multiple changes in DNA sequences and transcription factor balance must have occurred for the human gamma-globin gene to have evolved into a fetal gene. http://repub.eur.nl/res/pub/2461/ 1991-01-01T00:00:00Z We have used transgenic mice to study the influence of position of the human globin genes relative to the locus control region (LCR) on their expression pattern during development. The LCR, which is located 5' of the globin gene cluster, is normally required for the activation of all the genes. When the human beta-globin gene is linked as a single gene to the LCR it is activated prematurely in the embryonic yolk sac. We show that the correct timing of beta gene activation is restored when it is placed farther from the LCR than a competing human gamma- or alpha-globin gene. Correct timing is not restored when beta is the globin gene closest to the LCR. Similarly, the human gamma-globin gene is silenced earlier when present farthest from the LCR. On the basis of this result, we propose a model of developmental gene control based on stage-specific elements immediately flanking the genes and on polarity in the locus. We suggest that the difference in relative distance to the LCR, which is a consequence of the ordered arrangement of the genes, results in nonreciprocal competition between the genes for activation by the LCR. http://repub.eur.nl/res/pub/2462/ 1991-01-01T00:00:00Z Erythropoiesis during human development is characterized by switches in expression of beta-like globin genes during the transition from the embryonic through fetal to adult stages. Activation and high-level expression of the genes is directed by the locus control region (LCR), located 5' to the epsilon gene. The location of the LCR and its role in directing high-level expression of the globin genes has led to the suggestion that competition from the beta gene for interaction with the LCR has a major role in silencing the fetal gamma genes during adult life. We have now constructed lines of transgenic mice containing the human A gamma globin gene linked to the LCR. We observe high-level expression of the transgene in the embryonic stages but silencing of the gene in adult animals. We conclude that the gamma gene is not deregulated by the presence of the LCR and that competition from the beta gene is not required for silencing of the gamma genes in adult life. The silencing is therefore likely to be mediated by stage-specific factors binding to sequences immediately flanking the genes. http://repub.eur.nl/res/pub/2469/ 1991-01-01T00:00:00Z Transgenic mice were generated which carried the adult alpha and beta-globin genes and the major tadpole specific beta-globin gene of Xenopus laevis. The adult specific alpha and beta genes were found to express in erythroid tissues in adult mice, while the major tadpole specific beta gene (beta T1) was expressed in blood from 12.5 day embryos. The pattern of expression of the beta T1 gene during mouse development was consistent with its being regulated as an embryonic globin gene in the mouse. This observation suggests that some of the factors mediating globin switching have been conserved during the evolution of modern amphibia and mammals and raises interesting questions concerning the evolution of vertebrate globin gene switching. http://repub.eur.nl/res/pub/2470/ 1991-01-01T00:00:00Z