http://repub.eur.nl/res/aut/5624/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/26269/ 2011-04-01T00:00:00Z Neuroglobin (Ngb), a recently found oxygen-binding protein belonging to the vertebrate globin family, is mainly expressed in neurons of brains and eyes. Current studies have revealed diverse potential functions of Ngb and it was found to be able to reduce the severity of stroke and Alzheimer's disease, implying its importance in brains. However, the mechanism of Ngb regulation of transcription has not been elucidated yet. In this study, we analyzed the 5'-flanking region of human neuroglobin gene (NGB) and identified a transcription start site (TSS) located at -306. bp relative to the translation start site ATG. We characterized the proximal promoter of NGB and found two GC-boxes located at -16 and +. 30. bp relative to the TSS which are bound by transcription factor Sp1 and Sp3. Mutation of either GC-box led to a significant reduction in NGB promoter activity, while overexpression of Sp1 and Sp3 resulted in activation of the promoter. However, two putative NRSE sites (-359 and -127. bp relative to the TSS) apparently showed no influence on NGB tissue-specific expression. Treatment of two non-neuronal cell lines HeLa and BEAS-2B with 5-aza-2'-deoxycytidine remarkably induced NGB expression, suggesting a potential role of DNA methylation in regulating NGB tissue-specific expression. http://repub.eur.nl/res/pub/30528/ 2008-05-07T00:00:00Z Hypersensitive site 5 (5′HS5) of the β-globin Locus Control Region functions as a developmental stage-specific border in erythroid cells. Here, we have analyzed the role of 59HS5 in the three dimensional organization of the β-gene locus using the Chromatin Conformation Capture (3C) technique. The results show that when 5′HS5 is deleted from the locus, both remote and internal regulatory elements are still able to interact with each other in a three-dimensional configuration termed the Active Chromatin Hub. Thus, the absence of 5′HS5 does not have an appreciable effect on the three dimensional organization of the β-globin locus. This rules out models in which 5′HS5 nucleates interactions with remote and/or internal regulatory elements. We also determined the binding of CTCF, the only defined insulator protein in mammalian cells, to 5′HS5 by using chromatin immunoprecipitation (ChIP) assays. We detect low levels of CTCF binding to 5′HS5 in primitive erythroid cells, in which it functions as a border element. Surprisingly, we also observe binding levels of CTCF to 5′HS5 in definitive erythroid cells. Thus, binding of CTCF to 5′HS5 per se does not render it a functional border element. This is consistent with the previous data suggesting that CTCF has dual functionality. http://repub.eur.nl/res/pub/35053/ 2007-12-07T00:00:00Z Cytoglobin (CYGB) is ubiquitously expressed in all tissues and has been characterized as a respiratory protein in connective tissues. CYGB is up-regulated during hypoxia, implicating its function in maintaining the homeostasis redox of the cell. Here, we study the underlying molecular mechanisms by which hypoxia regulates human CYGB gene expression. When cells were subjected to hypoxia, the expression of endogenous CYGB was up-regulated ∼1.7-fold in BEAS-2B cells (p ≤ 0.05) and ∼1.6-fold in HeLa cells (p ≤ 0.05). Dual-luciferase assays and site directed mutagenesis showed the presence of hypoxia responsive elements (HREs) at positions -141, -144 and -448 that were essential for activation of CYGB expression under hypoxic conditions. The binding of hypoxia inducible factor (HIF-1) protein to the HREs was confirmed by gel shift and chromatin immunoprecipitation (ChIP) assays. These results indicate that HRE motifs are directly involved in the activation of the CYGB transcription under hypoxia. http://repub.eur.nl/res/pub/36093/ 2007-05-01T00:00:00Z In this report we examine the (AC)n(AT)xTymotif residing -530 bp 5′ upstream of the β-globin gene in Chinese thalassaemic patients. This motif is a putative binding site for a repressor protein, termed beta protein 1 (BP1) (Berg et al., Nucleic Acids Res 1989;17:8833-8852). Variations in the (AC)n(AT)xTyrepeats affect the binding affinity of BP1, thereby altering the expression of the β-globin gene. Eight different configurations of this repeat motif are identified in our population of Chinese β-thalassaemia patients. A (AC)3(AT)7T5motif was identified among these thalassaemia patients and its influence in β-globin gene expression was studied using stable transfection assay in murine erythroleukemia (MEL) cells. Our data demonstrated that the (AC)3(AT)7T5motif has a moderately strong repressor effect on the expression of the cis-linked β-globin gene. The high affinity of BP1 for this motif may result in the suppression of the transcription of the β-globin gene (Berg et al., Am J Hematol 1991;36:42-47). We postulate that silencer elements in the β-globin promoter play an important role in modifying the clinical presentation of the disease. 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/2527/ 1996-10-01T00:00:00Z Locus control regions (LCRs) are responsible for initiating and maintaining a stable tissue-specific open chromatin structure of a locus. In transgenic mice, LCRs confer high level expression on linked genes independent of position in the mouse genome. Here we show that an incomplete LCR loses this property when integrated into heterochromatic regions. Two disruption mechanisms were observed. One is classical position-effect variegation, resulting in continuous transcription in a clonal subpopulation of cells. The other is a novel mechanism resulting in intermittent gene transcription in all cells. We conclude that only a complete LCR fully overcomes heterochromatin silencing and that it controls the level of transcription by ensuring activity in all cells at all times rather than directly controlling the rate of transcription.