http://repub.eur.nl/res/aut/60/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/34359/ 2011-09-01T00:00:00Z Pluripotent stem cells exist in naive and primed states, epitomized by mouse embryonic stem cells (ESCs) and the developmentally more advanced epiblast stem cells (EpiSCs; ref.). In the naive state of ESCs, the genome has an unusual open conformation and possesses a minimum of repressive epigenetic marks. In contrast, EpiSCs have activated the epigenetic machinery that supports differentiation towards the embryonic cell types. The transition from naive to primed pluripotency therefore represents a pivotal event in cellular differentiation. But the signals that control this fundamental differentiation step remain unclear. We show here that paracrine and autocrine Wnt signals are essential self-renewal factors for ESCs, and are required to inhibit their differentiation into EpiSCs. Moreover, we find that Wnt proteins in combination with the cytokine LIF are sufficient to support ESC self-renewal in the absence of any undefined factors, and support the derivation of new ESC lines, including ones from non-permissive mouse strains. Our results not only demonstrate that Wnt signals regulate the naive-to-primed pluripotency transition, but also identify Wnt as an essential and limiting ESC self-renewal factor. http://repub.eur.nl/res/pub/33766/ 2011-06-08T00:00:00Z The POU domain transcription factor Pou3f1 (Oct6/Scip/Tst1) initiates the transition from ensheathing, promyelinating Schwann cells to myelinating cells. Axonal and other extracellular signals regulate Oct6 expression through the Oct6 Schwann cell enhancer (SCE), which is both required and sufficient to drive all aspects of Oct6 expression in Schwann cells. Thus, the Oct6 SCE is pivotal in the gene regulatory network that governs the onset of myelin formation in Schwann cells and provides a link between myelin promoting signaling and activation of a myelin-related transcriptional network. In this study, we define the relevant cis-acting elements within the SCE and identify the transcription factors that mediate Oct6 regulation. On the basis of phylogenetic comparisons and functional in vivo assays, we identify a number of highly conserved core elements within the mouse SCE. We show that core element 1 is absolutely required for full enhancer function and that it contains closely spaced inverted binding sites for Sox proteins. For the first time in vivo, the dimeric Sox10 binding to this element is shown to be essential for enhancer activity, whereas monomeric Sox10 binding is nonfunctional. As Oct6 and Sox10 synergize to activate the expression of the major myelin-related transcription factor Krox20, we propose that Sox10-dependent activation of Oct6 defines a feedforward regulatory module that serves to time and amplify the onset of myelination in the peripheral nervous system. http://repub.eur.nl/res/pub/27544/ 2010-09-01T00:00:00Z B-cell receptor (BCR)-mediated signals provide the basis for B-cell differentiation in the BM and subsequently into follicular, marginal zone, or B-1 B-cell subsets. We have previously shown that B-cell-specific expression of the constitutive active E41K mutant of the BCR-associated molecule Bruton's tyrosine kinase (Btk) leads to an almost complete deletion of immature B cells in the BM. Here, we report that low-level expression of the E41K or E41K-Y223F Btk mutants was associated with reduced follicular B-cell numbers and significantly increased proportions of B-1 cells in the spleen. Crosses with 3-83μδ and VH81X BCR Tg mice showed that constitutive active Btk expression did not change follicular, marginal zone, or B-1 B-cell fate choice, but resulted in selective expansion or survival of B-1 cells. Residual B cells were hyperresponsive and manifested sustained Ca2+mobilization. They were spontaneously driven into germinal center-independent plasma cell differentiation, as evidenced by increased numbers of IgM+plasma cells in spleen and BM and significantly elevated serum IgM. Because anti-nucleosome autoantibodies and glomerular IgM deposition were present, we conclude that constitutive Btk activation causes defective B-cell tolerance, emphasizing that Btk signals are essential for appropriate regulation of B-cell activation. http://repub.eur.nl/res/pub/23951/ 2010-01-01T00:00:00Z Germ line gene transposition technology has been used to generate "libraries" of flies and worms carrying genomewide mutations. Phenotypic screening and DNA sequencing of such libraries provide functional information resulting from insertional events in target genes. There is also a great need to have a fast and efficient way to generate mouse mutants in vivo to model developmental defects and human diseases. Here we describe an optimized mammalian germ line transposition system active during early mouse spermatogenesis using the Minos transposon. Transposon-positive progeny carry on average more than 2 new transpositions, and 45 to 100% of the progeny carry an insertion in a gene. The optimized Minos-based system was tested in a small rapid dominant functional screen to identify mutated genes likely to cause measurable cardiovascular "disease" phenotypes in progeny/embryos. Importantly this system allows rapid screening for modifier genes. Copyright http://repub.eur.nl/res/pub/25324/ 2009-11-18T00:00:00Z The simian virus 40 (SV40) T antigen is a potent oncogene able to transform many cell types and has been implicated in leukemia and lymphoma. In this report, we have achieved sporadic SV40 Tantigen expression in mature B cells in mice, by insertion of a SV40 T antigen gene in opposite transcriptional orientation in the immunoglobulin (Ig) heavy (H) chain locus between the D and JHsegments. SV40 T-antigen expression appeared to result from retention of the targeted germline allele and concomitant antisense transcription of SV40 large T in mature B cells, leading to chronic lymphocytic leukemia (CLL). Although B-cell development was unperturbed in young mice, aging mice showed accumulation of a monoclonal B-cell population in which the targeted IgH allele was in germline configuration and the wild-type IgH allele had a productive V(D)J recombination. These leukemic B cells were IgDlowCD5and manifested nonrandom usage of V, D, and J segments. VHregions were either unmutated, with preferential usage of the VH11 family, or manifested extensive somatic hypermutation. Our findings provide an animal model for B-CLL and show that pathways activated by SV40 T antigen play important roles in the pathogenesis of B-CLL. http://repub.eur.nl/res/pub/24972/ 2009-10-01T00:00:00Z Studies based on cell-free systems and on in vitro-cultured living cells support the concept that many cellular processes, such as transcription initiation, are highly dynamic: individual proteins stochastically bind to their substrates and disassemble after reaction completion. This dynamic nature allows quick adaptation of transcription to changing conditions. However, it is unknown to what extent this dynamic transcription organization holds for postmitotic cells embedded in mammalian tissue. To allow analysis of transcription initiation dynamics directly into living mammalian tissues, we created a knock-in mouse model expressing fluorescently tagged TFIIH. Surprisingly and in contrast to what has been observed in cultured and proliferating cells, postmitotic murine cells embedded in their tissue exhibit a strong and long-lasting transcription-dependent immobilization of TFIIH. This immobilization is both differentiation driven and development dependent. Furthermore, although very statically bound, TFIIH can be remobilized to respond to new transcriptional needs. This divergent spatiotemporal transcriptional organization in different cells of the soma revisits the generally accepted highly dynamic concept of the kinetic framework of transcription and shows how basic processes, such as transcription, can be organized in a fundamentally different fashion in intact organisms as previously deduced from in vitro studies. http://repub.eur.nl/res/pub/29259/ 2008-08-26T00:00:00Z Chronic stalling of DNA replication forks caused by DNA damage can lead to genomic instability. Cells have evolved lesion bypass pathways such as postreplication repair (PRR) to resolve these arrested forks. In yeast, one branch of PRR involves proliferating cell nuclear antigen (PCNA) polyubiquitination mediated by the Rad5-Ubc13-Mms2 complex that allows bypass of DNA lesion by a template-switching mechanism. Previously, we identified human SHPRH as a functional homologue of yeast Rad5 and revealed the existence of RAD5-like pathway in human cells. Here we report the identification of HLTF as a second RAD5 homologue in human cells. HLTF, like SHPRH, shares a unique domain architecture with Rad5 and promotes lysine 63-linked polyubiquitination of PCNA. Similar to yeast Rad5, HLTF is able to interact with UBC13 and PCNA, as well as SHPRH; and the reduction of either SHPRH or HLTF expression enhances spontaneous mutagenesis. Moreover, Hltf-deficient mouse embryonic fibroblasts show elevated chromosome breaks and fusions after methyl methane sulfonate treatment. Our results suggest that HLTF and SHPRH are functional homologues of yeast Rad5 that cooperatively mediate PCNA polyubiquitination and maintain genomic stability. http://repub.eur.nl/res/pub/36964/ 2007-11-01T00:00:00Z Faithful duplication of the genome requires structure-specific endonucleases such as the RuvABC complex in Escherichia coli. These enzymes help to resolve problems at replication forks that have been disrupted by DNA damage in the template. Much less is known about the identities of these enzymes in mammalian cells. Mus81 is the catalytic component of a eukaryotic structure-specific endonuclease that preferentially cleaves branched DNA substrates reminiscent of replication and recombination intermediates. Here we explore the mechanisms by which Mus81 maintains chromosomal stability. We found that Mus81 is involved in the formation of double-strand DNA breaks in response to the inhibition of replication. Moreover, in the absence of chromosome processing by Mus81, recovery of stalled DNA replication forks is attenuated and chromosomal aberrations arise. We suggest that Mus81 suppresses chromosomal instability by converting potentially detrimental replication-associated DNA structures into intermediates that are more amenable to DNA repair. http://repub.eur.nl/res/pub/36584/ 2007-09-21T00:00:00Z The pre-B cell receptor (pre-BCR), composed of immunoglobulin μ heavy chain and the surrogate light chain (SLC) proteins λ5 and Vpreb, signals for proliferation and maturation of developing pre-B cells. It has been assumed that pre-B cells stop cycling by the pre-BCR-mediated downregulation of SLC transcription. We generated transgenic mice expressing SLC throughout B cell development and, remarkably, found that enforced SLC expression had no effect on pre-B cell proliferation or differentiation. However, in the presence of conventional immunoglobulin light chains, SLC components had the capacity to induce constitutive BCR internalization, secondary immunoglobulin light-chain rearrangement, and a severe developmental arrest of immature B cells, dependent on the adaptor protein Slp65. Residual B cells in the spleen showed increased expression of surface CD5, which is a negative regulator of BCR signaling, and differentiated spontaneously into IgM+plasma cells. Thus, the silencing of SLC genes is not essential for the limitation of pre-B cell proliferation, but is required for the prevention of constitutive activation of B cells. http://repub.eur.nl/res/pub/35568/ 2007-03-01T00:00:00Z The Fibulins are a 6-member protein family hypothesized to function as intermolecular bridges that stabilize the organization of extracellular matrix structures. Here, we show that reduced expression of Fibulin-4 leads to aneurysm formation, dissection of the aortic wall and cardiac abnormalities. Fibulin-4 knockdown mice with a hypomorphic expression allele arose from targeted disruption of the adjacent Mus81 endonuclease gene. Mice homozygous for the Fibulin-4 reduced expression allele (Fibulin-4) show dilatation of the ascending aorta and a tortuous and stiffened aorta, resulting from disorganized elastic fiber networks. They display thickened aortic valvular leaflets that are associated with aortic valve stenosis and insufficiency. Strikingly, already a modest reduction in expression of Fibulin-4 in the heterozygous Fibulin-4 mice occasionally resulted in small aneurysm formation. To get insight into the underlying molecular pathways involved in aneurysm formation and response to aortic failure, we determined the aorta transcriptome of Fibulin-4 and Fibulin-4 animals and identified distinct and overlapping biological processes that were significantly overrepresented including cytoskeleton organization, cell adhesion, apoptosis and several novel gene targets. Transcriptome and protein expression analysis implicated perturbation of TGF-β signaling in the pathogenesis of aneurysm in fibulin-4 deficient mice. Our results show that the dosage of a single gene can determine the severity of aneurysm formation and imply that disturbed TGF-β signaling underlies multiple aneurysm phenotypes. http://repub.eur.nl/res/pub/13479/ 2004-09-01T00:00:00Z Genetic defects in DNA repair mechanisms and cell cycle checkpoint (CCC) genes result in increased genomic instability and cancer predisposition. Discovery of mammalian homologs of yeast CCC genes suggests conservation of checkpoint mechanisms between yeast and mammals. However, the role of many CCC genes in higher eukaryotes remains elusive. Here, we report that targeted deletion of an N-terminal part of mRad17, the mouse homolog of the Schizosaccharomyces pombe Rad17 checkpoint clamp-loader component, resulted in embryonic lethality during early/mid-gestation. In contrast to mouse embryos, embryonic stem (ES) cells, isolated from mRad17(5'Delta/5'Delta) embryos, produced truncated mRad17 and were viable. These cells displayed hypersensitivity to various DNA-damaging agents. Surprisingly, mRad17(5'Delta/5'Delta) ES cells were able to arrest cell cycle progression upon induction of DNA damage. However, they displayed impaired homologous recombination as evidenced by a strongly reduced gene targeting efficiency. In addition to a possible role in DNA damage-induced CCC, based on sequence homology, our results indicate that mRad17 has a function in DNA damage-dependent recombination that may be responsible for the sensitivity to DNA-damaging agents. http://repub.eur.nl/res/pub/3222/ 2004-01-01T00:00:00Z Interstrand cross-links (ICLs) are an extremely toxic class of DNA damage incurred during normal metabolism or cancer chemotherapy. ICLs covalently tether both strands of duplex DNA, preventing the strand unwinding that is essential for polymerase access. The mechanism of ICL repair in mammalian cells is poorly understood. However, genetic data implicate the Ercc1-Xpf endonuclease and proteins required for homologous recombination-mediated double-strand break (DSB) repair. To examine the role of Ercc1-Xpf in ICL repair, we monitored the phosphorylation of histone variant H2AX (gamma-H2AX). The phosphoprotein accumulates at DSBs, forming foci that can be detected by immunostaining. Treatment of wild-type cells with mitomycin C (MMC) induced gamma-H2AX foci and increased the amount of DSBs detected by pulsed-field gel electrophoresis. Surprisingly, gamma-H2AX foci were also induced in Ercc1(-/-) cells by MMC treatment. Thus, DSBs occur after cross-link damage via an Ercc1-independent mechanism. Instead, ICL-induced DSB formation required cell cycle progression into S phase, suggesting that DSBs are an intermediate of ICL repair that form during DNA replication. In Ercc1(-/-) cells, MMC-induced gamma-H2AX foci persisted at least 48 h longer than in wild-type cells, demonstrating that Ercc1 is required for the resolution of cross-link-induced DSBs. MMC triggered sister chromatid exchanges in wild-type cells but chromatid fusions in Ercc1(-/-) and Xpf mutant cells, indicating that in their absence, repair of DSBs is prevented. Collectively, these data support a role for Ercc1-Xpf in processing ICL-induced DSBs so that these cytotoxic intermediates can be repaired by homologous recombination. http://repub.eur.nl/res/pub/10244/ 2003-01-01T00:00:00Z The Tec family member Bruton's tyrosine kinase (Btk) is a cytoplasmic protein tyrosine kinase that transduces signals from the pre-B and B cell receptor (BCR). Btk is involved in pre-B cell maturation by regulating IL-7 responsiveness, cell surface phenotype changes, and the activation of lambda L chain gene rearrangements. In mature B cells, Btk is essential for BCR-mediated proliferation and survival. Upon BCR stimulation, Btk is transphosphorylated at position Y551, which promotes its catalytic activity and subsequently results in autophosphorylation at position Y223 in the Src homology 3 domain. To address the significance of Y223 autophosphorylation and the requirement of enzymatic activity for Btk function in vivo, we generated transgenic mice that express the autophosphorylation site mutant Y223F and the kinase-inactive mutant K430R, respectively. We found that Y223 autophosphorylation was not required for the regulation of IL-7 responsiveness and cell surface phenotype changes in differentiating pre-B cells, or for peripheral B cell differentiation. However, expression of the Y223F-Btk transgene could not fully rescue the reduction of lambda L chain usage in Btk-deficient mice. In contrast, transgenic expression of kinase-inactive K430R-Btk completely reconstituted lambda usage in Btk-deficient mice, but the defective modulation of pre-B cell surface markers, peripheral B cell survival, and BCR-mediated NF-kappaB induction were partially corrected. From these findings, we conclude that: 1) autophosphorylation at position Y223 is not essential for Btk function in vivo, except for regulation of lambda L chain usage, and 2) during B cell development, Btk partially acts as an adapter molecule, independent of its catalytic activity. http://repub.eur.nl/res/pub/9639/ 2001-01-01T00:00:00Z Bruton's tyrosine kinase (Btk) is a nonreceptor tyrosine kinase involved in precursor B (pre-B) cell receptor signaling. Here we demonstrate that Btk-deficient mice have an approximately 50% reduction in the frequency of immunoglobulin (Ig) lambda light chain expression, already at the immature B cell stage in the bone marrow. Conversely, transgenic mice expressing the activated mutant Btk(E41K) showed increased lambda usage. As the kappa/lambda ratio is dependent on (a) the level and kinetics of kappa and lambda locus activation, (b) the life span of pre-B cells, and (c) the extent of receptor editing, we analyzed the role of Btk in these processes. Enforced expression of the Bcl-2 apoptosis inhibitor did not alter the Btk dependence of lambda usage. Crossing 3-83mudelta autoantibody transgenic mice into Btk-deficient mice showed that Btk is not essential for receptor editing. Also, Btk-deficient surface Ig(+) B cells that were generated in vitro in interleukin 7-driven bone marrow cultures manifested reduced lambda usage. An intrinsic defect in lambda locus recombination was further supported by the finding in Btk-deficient mice of reduced lambda usage in the fraction of pre-B cells that express light chains in their cytoplasm. These results implicate Btk in the regulation of the activation of the lambda locus for V(D)J recombination in pre-B cells. http://repub.eur.nl/res/pub/17508/ 1998-12-16T00:00:00Z Each individual organism has to protect itself against a large variety of infectious microbial agents, such as bacteria, fungi and parasites to prevent pathological damage and death. In vertebrates, defense mechanisms against foreign substances, antigens, have evolved in the immune system, which has two functional divisions: the 'innate' immune system and the 'adaptive' immune system. The 'innate' immune system is aspecific and acts as a first line of defense, mediated by cells from thc myeloid lineage and soluble factors like complement and lysozyme. The main function of the 'innate' immune system is to avoid entering of micro or gall isms into the body and to clear it of killed pathogens. In contrast to the 'adaptive' immune system, repeated infection does not improve the resistance of the 'innate' immune system. If the first line of defense is defeated, the second line of defense, the 'adaptive' immune system, which is very specific and can develop memory to earlier accounted pathogens, is activated. The specific immune response is mediated by lymphocytes belonging to the B andlor T lineages (B and T cells). Both Band T cells express receptor molecules on their cell membrane, which specifically can bind antigens. The T ceH receptor (TCR) can only bind antigens if these are processed into small peptides, and presented by major histocompatibility complex (MHC) class I molecules on the surface of host cells and MHC class II molecules on the surface of antigen presenting cells. Intracellular antigens are processed into small peptides and presented on the surface by the MHC class I complex. Recognition of the MHC-class I-peptide complex by the TCR of cytotoxic T cells results in killing of the presenting host cells. MHC class II molecules present processed peptides, which are derived from external clldocytosed antigens. Recognition of MHC class II-peptide complexes by the TCR ofT helper (T H) cells results in the production of cytokines and stimulation of cells of the immune system ('cellular' immune response). The B cell receptor (BCR) binds to unprocessed antigens. Stimulation of the BCR results in a 'humoral' immune response, i.e. the secretion of soluble immunoglobulins (Ig)J with the same binding specificity as the BCR to the triggering antigen. http://repub.eur.nl/res/pub/12802/ 1998-09-15T00:00:00Z To identify B-cell signaling pathways activated by Bruton's tyrosine kinase (Btk) in vivo, we generated transgenic mice in which Btk expression is driven by the MHC class II Ea gene locus control region. Btk overexpression did not have significant adverse effects on B cell function, and essentially corrected the X-linked immunodeficiency (xid) phenotype in Btk- mice. In contrast, expression of a constitutively activated form of Btk carrying the E41K gain-of-function mutation resulted in a B cell defect that was more severe than xid. The mice showed a marked reduction of the B cell compartment in spleen, lymph nodes, peripheral blood and peritoneal cavity. The levels in the serum of most immunoglobulin subclasses decreased with age, and B cell responses to both T cell-independent type II and T cell-dependent antigens were essentially absent. Expression of the E41K Btk mutant enhanced blast formation of splenic B cells in vitro in response to anti-IgM stimulation. Furthermore, the mice manifested a disorganization of B cell areas and marginal zones in the spleen. Our findings demonstrate that expression of constitutively activated Btk blocks the development of follicular recirculating B cells. http://repub.eur.nl/res/pub/2548/ 1997-01-01T00:00:00Z http://repub.eur.nl/res/pub/4462/ 1992-01-01T00:00:00Z The effects of intravenous elgodipine, a new second-generation dihydropyridine calcium antagonist, on hemodynamics and coronary artery diameter were investigated in 15 patients undergoing cardiac catheterization for suspected coronary artery disease. Despite a significant decrease in systemic blood pressure, elgodipine infused at a rate of 1.5 micrograms/kg/min over a period of 10 minutes did not affect heart rate and left ventricular end-diastolic pressure. The contractile responses during isovolumic contraction showed a slight but significant increase in maximum velocity (56 +/- 10 to 60 +/- 10 seconds-1; p less than 0.005), whereas the time constant of early relaxation was shortened from 49 +/- 11 to 44 +/- 9 ms (p less than 0.05). Coronary sinus and great cardiac vein flow increased significantly by 15 and 26%, respectively. As mean aortic pressure decreased, a significant decrease in coronary sinus (-27%) and great cardiac vein (-28%) resistance was observed, while the calculated myocardial oxygen consumption remained unchanged. In all, 69 coronary segments (including 13 stenotic segments) were analyzed quantitatively using computer-assisted quantitative coronary angiography. A significant increase in mean coronary artery diameter (2.27 +/- 0.53 to 2.48 +/- 0.53 mm; p less than 0.000001), as well as in obstruction diameter, (1.08 +/- 0.29 to 1.36 +/- 0.32 mm; p less than 0.02), was observed. The results demonstrate that elgodipine, in the route and dose described, induces significant vasodilatation of both coronary resistance and epicardial conductance vessels, without adverse effects on heart rate, myocardial oxygen demand and contractile indexes.