http://repub.eur.nl/res/aut/724/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/33286/ 2011-09-27T00:00:00Z Panton-Valentine leukocidin (PVL) is a pore-forming toxin associated with current outbreaks of community-associated methicillin-resistant strains and implicated directly in the pathophysiology of Staphylococcus aureus-related diseases. Humanized heavy chain-only antibodies (HCAb) were generated against S. aureus PVL from immunized transgenicmice to neutralize toxin activity. The active form of PVL consists of the two components, LukS-PV and LukF-PV, which induce osmotic lysis following pore formation in host defense cells. One anti-LukS-PV HCAb, three anti-LukF-PV HCAbs with affinities in the nanomolar range, and one engineered tetravalent bispecific HCAb were tested in vitro and in vivo, and all prevented toxin binding and pore formation. Anti-LukS-PV HCAb also binds to γ-hemolysin C (HlgC) and inhibits HlgC/HlgB pore formation. Experiments in vivo in a toxin-induced rabbit endophthalmitis model showed that these HCAbs inhibit inflammatory reactions and tissue destruction, with the tetravalent bispecific HCAb performing best. Our findings show the therapeutic potential of HCAbs, and in particular, bispecific antibodies. http://repub.eur.nl/res/pub/28059/ 2010-02-01T00:00:00Z One of the complexes formed by the hematopoietic transcription factor Gata1 is a complex with the Ldb1 (LIM domain-binding protein 1) and Tal1 proteins. It is known to be important for the development and differentiation of the erythroid cell lineage and is thought to be implicated in long-range interactions. Here, the dynamics of the composition of the complex - in particular, the binding of the negative regulators Eto2 and Mtgr1 - are studied, in the context of their genome-wide targets. This shows that the complex acts almost exclusively as an activator, binding a very specific combination of sequences, with a positioning relative to transcription start site, depending on the type of the core promoter. The activation is accompanied by a net decrease in the relative binding of Eto2 and Mtgr1. A Chromosome Conformation Capture sequencing (3C-seq) assay also shows that the binding of the Ldb1 complex marks genomic interaction sites in vivo. This establishes the Ldb1 complex as a positive regulator of the final steps of erythroid differentiation that acts through the shedding of negative regulators and the active interaction between regulatory sequences. http://repub.eur.nl/res/pub/24942/ 2009-02-05T00:00:00Z Background: Chromatin immunoprecipitation (ChIP) assays coupled to genome arrays (Chip-on-chip) or massive parallel sequencing (ChIP-seq) lead to the genome wide identification of binding sites of chromatin associated proteins. However, the highly variable quality of antibodies and the availability of epitopes in crosslinked chromatin can compromise genomic ChIP outcomes. Epitope tags have often been used as more reliable alternatives. In addition, we have employed protein in vivo biotinylation tagging as a very high affinity alternative to antibodies. In this paper we describe the optimization of biotinylation tagging for ChIP and its coupling to a known epitope tag in providing a reliable and efficient alternative to antibodies. Results: Using the biotin tagged erythroid transcription factor GATA-1 as example, we describe several optimization steps for the application of the high affinity biotin streptavidin system in ChIP. We find that the omission of SDS during sonication, the use of fish skin gelatin as blocking agent and choice of streptavidin beads can lead to significantly improved ChIP enrichments and lower background compared to antibodies. We also show that the V5 epitope tag performs equally well under the conditions worked out for streptavidin ChIP and that it may suffer less from the effects of formaldehyde crosslinking. Conclusion: The combined use of the very high affinity biotin tag with the less sensitive to crosslinking V5 tag provides for a flexible ChIP platform with potential implications in ChIP sequencing outcomes. http://repub.eur.nl/res/pub/13816/ 2005-07-06T00:00:00Z GATA-1 is essential for the generation of the erythroid, megakaryocytic, eosinophilic and mast cell lineages. It acts as an activator and repressor of different target genes, for example, in erythroid cells it represses cell proliferation and early hematopoietic genes while activating erythroid genes, yet it is not clear how both of these functions are mediated. Using a biotinylation tagging/proteomics approach in erythroid cells, we describe distinct GATA-1 interactions with the essential hematopoietic factor Gfi-1b, the repressive MeCP1 complex and the chromatin remodeling ACF/WCRF complex, in addition to the known GATA-1/FOG-1 and GATA-1/TAL-1 complexes. Importantly, we show that FOG-1 mediates GATA-1 interactions with the MeCP1 complex, thus providing an explanation for the overlapping functions of these two factors in erythropoiesis. We also show that subsets of GATA-1 gene targets are bound in vivo by distinct complexes, thus linking specific GATA-1 partners to distinct aspects of its functions. Based on these findings, we suggest a model for the different roles of GATA-1 in erythroid differentiation. 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/13165/ 2003-06-24T00:00:00Z Proteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin/streptavidin for biotinylated templates. Here, we describe an approach for the single-step purification of transcription factor complex(es) based on specific in vivo biotinylation. We expressed the bacterial BirA biotin ligase in mammalian cells and demonstrated very efficient biotinylation of a hematopoietic transcription factor bearing a small (23-aa) artificial peptide tag. Biotinylation of the tagged transcription factor altered neither the factor's protein interactions or DNA binding properties in vivo nor its subnuclear distribution. Using this approach, we isolated the biotin-tagged transcription factor and at least one other known interacting protein from crude nuclear extracts by direct binding to streptavidin beads. Finally, this method works efficiently in transgenic mice, thus raising the prospect of using biotinylation tagging in protein complex purification directly from animal tissues. Therefore, BirA-mediated biotinylation of tagged proteins provides the basis for the single-step purification of proteins from mammalian cells. http://repub.eur.nl/res/pub/12805/ 1998-10-15T00:00:00Z We have used a kinetic analysis to distinguish possible mechanisms of activation of transcription of the different genes in the human beta globin locus. Based on in situ studies at the single-cell level we have previously suggested a dynamic mechanism of single genes alternately interacting with the locus control region (LCR) to activate transcription. However, those steady-state experiments did not allow a direct measurement of the dynamics of the mechanism and the presence of loci with in situ primary transcript signals from two beta-like genes in cis has left open the possibility that multiple genes in the locus could initiate transcription simultaneously. Kinetic assays involving removal of a block to transcription elongation in conjunction with RNA FISH show that multiple beta gene primary transcript signals in cis represent a transition between alternating transcriptional periods of single genes, supporting a dynamic interaction mechanism. 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. http://repub.eur.nl/res/pub/2520/ 1995-01-01T00:00:00Z DNA-protein interaction studies in vitro revealed several factors binding over the TATA box and the region of transcription initiation (cap) site of the human beta-globin promoter; TATA binding protein TBP at -30, Sp1 at -19, GATA-1 at -12 and +5, YY1 at -9 and a novel factor C1 over the site of initiation (-4 to +7). Point mutants which specifically abolish the binding of each of these proteins were tested in a beta-globin locus control region (LCR) construct which allows quantitative comparisons at physiological levels of transcription. Only mutants which drastically affect the binding of TBP resulted in decreased levels of transcription. A threshold value of TBP binding of 15-30% of wild type was sufficient to give normal levels of transcription. This indicates that the association of TF IID with the TATA box is not limiting in the rate of initiation of transcription. http://repub.eur.nl/res/pub/2434/ 1989-01-01T00:00:00Z We have studied the erythroid-specific promoter of the human gene coding for Porphobilinogen Deaminase (PBGD) by DNaseI footprinting, gel retardation and methylation interference assays. We show that this promoter, which is inducible during MEL cell differentiation, contains three binding sites for the erythroid-specific factor NF-E1 and one site for a second erythroid-specific factor, which we name NF-E2. NF-E1 is a factor that also binds the promoter and the enhancer (present in the 3' flanking region) of the human beta-globin gene. NF-E2 has not yet been described and although it binds to a sequence containing the Ap1 consensus, it appears to be different from Ap1. http://repub.eur.nl/res/pub/2436/ 1989-01-01T00:00:00Z We have used DNaseI footprinting and gel mobility assays to analyze the upstream region of the human A gamma-globin gene promoter. Four protein factors were found to bind this region. A non-erythroid factor present in the 0.4M KCl fraction of a heparin agarose column binds to the CAC box (-140). A ubiquitous octamer factor present in the 0.2M fraction binds to an ATGCAAT element (-175), but is completed out by the erythroid specific factor NF-E1 (in the 0.4M KCl fraction), which binds a site (-186) immediately flanking the octamer. A novel factor binding to a stretch of 8A around -233, was identified in the 0.2M KCl fraction. This factor is not present in HeLa nuclear extracts. To study the transcriptional importance of these protein binding sites we have used an "A gamma-minilocus", similar to that described for the beta-globin gene (1) in K562 cells. This provides evidence that the NF-E1 and CAC box in the -210 to -122 region of the A gamma-promoter are important for the efficient expression of the gamma-globin gene. http://repub.eur.nl/res/pub/2426/ 1988-01-01T00:00:00Z We have introduced into murine erythroleukaemia (MEL) cells several series of deletion mutants of hybrid genes between the human beta-globin gene and the murine H-2Kb gene. S1 nuclease and transcriptional run-off analysis showed that the human beta-globin gene contains at least three globin specific transcriptional control elements. One is a promoter element and is located 160 bp upstream from the transcription initiation site; it increases the efficiency of the promoter after MEL cells are induced to differentiate. The other two elements are tissue-specific transcriptional enhancers and are located downstream from the transcription initiation site, the first in the structural beta-globin gene and the second in the 3' flanking sequences. http://repub.eur.nl/res/pub/2431/ 1988-01-01T00:00:00Z We have shown that the minimal enhancer fragment present in the 3'-flanking region of the human beta-globin gene contains four regions that bind nuclear proteins in vitro. By using gel mobility shift and DNase I footprinting assays, we were able to show that each of these regions binds an erythroid-cell-specific nuclear factor which we name NF-E1. This factor is present in erythroid cells at different developmental stages of globin gene expression. The recognition sequence of this protein (A/C Py T/A ATC A/T Py) is also present in the intragenic enhancer and the promoter of the beta-globin gene as well as in the promoter of other erythroid-cell specific genes. In addition to NF-E1, each of the four binding regions interacts with at least one other protein factor. http://repub.eur.nl/res/pub/2432/ 1988-01-01T00:00:00Z We have shown that the promoter of the human beta-globin gene contains three regions in addition to the known CAC, CAAT and TATA box regions that are important for the induction of transcription in erythroid cells. By using DNaseI footprinting and gel mobility shift assays we were able to show that two of these regions bind the erythroid specific nuclear factor NF-E1 (and ubiquitous factors). The third region binds a ubiquitous CAAT-box factor (CP1). Deletion experiments suggest that only the combination of NF-E1 and CP1 binding sites, but not each of the sites alone, are capable of mediating the induction of transcription of a minimal (CAC, CAAT, TATA box) beta-globin promoter in mouse erythroleukaemia (MEL) cells. http://repub.eur.nl/res/pub/2406/ 1987-01-01T00:00:00Z We report here the localisation of sequences responsible for the faulty expression of human beta-globin gene in Putko and K562 cells. Complete beta-globin gene introduced into these cells produces transcripts with abnormal 5' ends, while cotransfected mouse H2 gene is expressed correctly. Using hybrid constructs of these two genes we demonstrate that aberrant activity is conferred by sequences 5' of the beta-globin gene. Thus beta-globin promoter attached to the H2 coding sequence produces H2 transcripts with truncated 5' ends. By introducing a series of deletions in the beta-globin promoter we restrict these sequences to the -77/+28 base pair region spanning the CAAT element to the translation initiation site. These results are consistent with the lack of recognition of the beta-globin gene major cap site in Putko and K562 cells. We suggest that inactivity of the adult globin gene in the embryonic/fetal environment is at least in part conferred by sequences within the beta-globin gene promoter. http://repub.eur.nl/res/pub/2410/ 1987-01-01T00:00:00Z http://repub.eur.nl/res/pub/2418/ 1987-01-01T00:00:00Z The human P-globin gene is part of a multigene family and is expressed specifically in adult human erythroid tissue (for review, 1). When the human P-globin is introduced into fertilized mouse eggs, it is first activated in foetal liver and remains expressed in adult erythroid tissues (2,3,4). It therefore mimicks the pattern of expression of its murine counterpart. It has previously been shown in tissue culture (5) and transgenic mice (4) that sequences downstream from the 0-globin promoter are involved in this regulation. We now show that at least part of these sequences are located 0.5-1.2kb downstream from the polyA addition site and constitute a transcriptional enhancer element that is erythroid and developmental specific. http://repub.eur.nl/res/pub/2376/ 1984-01-01T00:00:00Z We have introduced into murine erythroleukaemia (MEL) cells a series of human globin gene cosmids and two sets of hybrid genes constructed from the human beta-globin gene and the human gamma-globin or murine H-2Kbm1 genes. S1-nuclease analysis of the mRNA products from these genes before and after MEL cell differentiation showed that the human beta-globin gene, but not the human epsilon- or gamma-globin or H-2Kbm1 genes, is induced specifically. Hybrid genes containing human beta-globin DNA sequences from either 5' or 3' side of the translation initiation site were both inducible. Measurement of the relative rate of transcription showed this induction to be the result of transcriptional activation. We therefore suggest that DNA sequences which regulate beta-globin gene expression during MEL differentiation are located both 5' and 3' to the translation initiation site. http://repub.eur.nl/res/pub/2356/ 1983-01-01T00:00:00Z http://repub.eur.nl/res/pub/2366/ 1983-01-01T00:00:00Z MspI essentially fails to cut the sequence GGCmCGG at enzyme concentrations which give total digestion of CCGG, CmCGG and GGCCGG sites. This result explains why certain sites in mammalian DNA are resistant to both MspI and HpaII and shows that this results from an idiosynchracy of MspI rather than a novel form of DNA methylation at this site in mammalian cells. http://repub.eur.nl/res/pub/2367/ 1983-01-01T00:00:00Z Chemically induced differentiation of cultured murine erythroleukaemia (MEL) cells results in a several hundred-fold increase in transcription of the adult mouse globin genes and thus serves as a model for gene activation during erythropoiesis. One approach to study gene regulation in this system has been to analyse the expression of foreign globin genes introduced into MEL cells. By introducing cosmid DNA containing the human adult(beta), fetal(gamma) and embryonic(epsilon)-globin genes, we have shown here that expression of the beta, but not the gamma or epsilon genes, is regulated during MEL differentiation. Regulated expression of the human beta-globin gene was observed when it was introduced either as part of the intact globin gene cluster or as an individual gene with 1.5 kilobases (kb) of 5' flanking DNA. Transcription from a herpes simplex virus (HSV) promoter adjacent to the thymidine kinase (tk) gene is also inducible in MEL cells. http://repub.eur.nl/res/pub/2350/ 1982-01-01T00:00:00Z http://repub.eur.nl/res/pub/2353/ 1982-01-01T00:00:00Z http://repub.eur.nl/res/pub/2342/ 1981-04-01T00:00:00Z A human gene library was constructed using an improved cloning technique for cosmid vectors. Human placental DNA was partially digested with restriction endonuclease MboI; size-fractionated and ligated to BamHI-cut and phosphatase-treated cosmid vector pJB8. After packaging in lambda phage particles, the recombinant DNA was transduced into Escherichia coli 1400 or HB101 followed by selection on ampicillin for recombinant E. coli. 150 000 recombinant-DNA-containing colonies were screened for the presence of the human beta-globin related genes. Five recombinants were isolated containing the human beta-globin locus and encompassing approx. 70 kb of human DNA. http://repub.eur.nl/res/pub/2347/ 1981-01-01T00:00:00Z We have cloned the single beta-globin gene from an Italian patient who is a double heterozygote for beta o/delta beta o thalassaemia. RNA isolated from nucleated red cells from this patient can be translated in vitro to give detectable levels of A gamma- G gamma and alpha-globin, but no beta-globin. S1-mapping transcription studies show that beta-globin mRNA is present at about 1-3% of the level of alpha- and gamma-globin mRNA. In addition, the expression of this gene has been studied by reversed genetics. SV40-plasmid-beta o-globin gene recombinants have been transfected into Hela cells and analysed for beta-globin mRNA. In contrast to the results obtained with mRNA isolated directly from the blood of this patient, in the transfected Hela cells the same level of beta-globin mRNA is seen for the beta o thalassaemic globin gene and for a normal beta-globin gene. To elucidate the nature of the lesion, the entire DNA sequence of the beta-globin gene of this patient has been determined. The sequence shows that this gene contains a termination codon at position 39 (CAG - greater than UAG). Otherwise, there is a remarkable conservation of the entire DNA sequence.