Biomedical subjects

F Grosveld

Publications and source records attributed to F Grosveld.

At least 73 records · Page 4Linked to original sources

PubMed1998

Locus control regions, chromatin activation and transcription.

The past year has seen interesting advances in our understanding of the action of locus control regions. For the first time, the chromosomal distance was described in detail as a parameter in positive/negative regulation of transcription via gene competition. A number of publications have also described negative regulatory elements which restrict the action of locus control regions and other regulatory regions to specific genes and/or specific tissues. The emerging picture indicates that several very different types of negative regulation ensure that transcriptional activation occurs only in the appropriate cells.

Animals↗

PubMed1998

The dynamics of globin gene expression and gene therapy vectors.

The most important level of the regulation of the beta-globin genes is by activation of all of the genes by the locus control region (LCR). Part of the developmental regulation of the locus is achieved by competition of the genes for the interaction with the LCR. Although this level of gene regulation is quantitatively of less importance than the direct repression mechanism for the early genes, it has important implications and has provided an excellent assay to probe the regulation of transcription at the single cell level. The results of these studies indicate that the LCR interacts with individual globin genes and that LCR/gene interactions are dynamic with complexes forming and dissociating continually. We conclude that transcription only appears to take place while the LCR and gene interact and that the level of transcription is determined by the frequency and duration of such interaction rather than by changes in the rate of transcription of the promoters. This mechanism has clear implications for the design of vectors for the purpose of gene therapy.

Animals↗

PubMed1997

Temporal and spatial control of murine GATA-3 transcription by promoter-proximal regulatory elements.

GATA-3 is expressed in a temporally dynamic manner and fulfills vital functions during vertebrate fetal development. Homozygous mGATA-3 mutant embryos die at midgestation, thus complicating the analysis of its contribution to the development of specific cell fates in the many tissues where it is expressed during embryogenesis. We show here that the elements controlling GATA-3 regulation can be precisely refined, using transgenic mice, to discrete cis-acting domains: within 6 kb surrounding the transcriptional initiation site, separate sequences were found to control the expression of mGATA-3 in early muscle masses, in a subset of PNS neurons, in the genital tubercle, and in the branchial arches. The branchial arch regulatory element is particularly robust and was refined to a discrete enhancer sequence lying between nt -2832 and -2462 from the transcription initiation site. The enhancer contains potential binding sites for many well-characterized transcription factors, suggesting that mGATA-3 transcriptional activity may be regulated by these proteins (or related family members) in the mesenchyme of the arches that contribute to formation of the jaw. These studies show that discrete regulatory elements required for the elaboration of complex developmental programs can be individually localized, suggesting that the developmentally transient expression of individual transcription factors collaboratively contributes to the temporal and spatial pattern of cellular differentiation leading to the formation of adult anatomy.

Animals↗

PubMed1997

Transcription factor Sp1 is essential for early embryonic development but dispensable for cell growth and differentiation.

Transcription factor Sp1 has been implicated in the expression of many genes. Moreover, it has been suggested that Sp1 is linked to the maintenance of methylation-free CpG islands, the cell cycle, and the formation of active chromatin structures. We have inactivated the mouse Sp1 gene. Sp1-/- embryos are retarded in development, show a broad range of abnormalities, and die around day 11 of gestation. In Sp1-/- embryos, the expression of many putative target genes, including cell cycle-regulated genes, is not affected, CpG islands remain methylation free, and active chromatin is formed at the globin loci. However, the expression of the methyl-CpG-binding protein MeCP2 is greatly reduced in Sp1-/- embryos. MeCP2 is thought to be required for the maintenance of differentiated cells. We suggest that Sp1 is an important regulator of this process.

Animals↗

PubMed1997

Evaluation of beta-globin gene therapy constructs in single copy transgenic mice.

Effective gene therapy constructs based on retrovirus or adeno-associated virus vectors will require regulatory elements that direct expression of genes transduced at single copy. Most beta-globin constructs designed for therapy of beta-thalassemias are regulated by the 5'HS2 component of the locus control region (LCR). Here we show that a human beta-globin gene flanked by two small 5'HS2 core elements or flanked by a 5'HS3 (footprints 1-3) core and a 5'HS2 core are not reproducibly expressed in single copy transgenic mice. In addition, low copy transgene concatamers that contain only dimer 5'HS2 cores fail to express, whereas those that contain monomer 5'HS2 cores express at 14% per copy. These data suggest that spacing between HS cores is crucial for LCR activity. We therefore constructed a novel 3.0 kb LCR cassette in which the 5'HS2, 5'HS3 and 5'HS4 cores are each separated by approximately 700 bp. When linked to the 815 bp beta-globin promoter this LCR directs 45% levels of expression from four independent single copy transgenes. However, the 3.0 kb LCR linked to the 265 bp promoter expresses variable levels, averaging 18%, from three single copy transgenes. Our findings suggest that sequences in the distal promoter play a role in single copy transgene activation and that larger LCR and promoter elements are most suitable for gene therapy applications.

Animals↗

PubMed1997

Correction of the X-linked immunodeficiency phenotype by transgenic expression of human Bruton tyrosine kinase under the control of the class II major histocompatibility complex Ea locus control region.

Bruton tyrosine kinase (Btk) is essential for the development of pre-B cells to mature B cell stages. Btk-deficient mice manifest an X-linked immunodeficiency (xid) defect characterized by a reduction of peripheral IgMlow IgDhigh B cells, a lack of peritoneal CD5+ B cells, low serum levels of IgM and IgG3, and impaired responses to T cell independent type II (TI-II) antigens. We have generated transgenic mice in which expression of the human Btk gene is driven by the murine class II major histocompatibility complex Ea gene locus control region, which provides gene expression from the pre-B cell stage onwards. When these transgenic mice were mated onto a Btk- background, correction of the xid B cell defects was observed: B cells differentiated to mature IgMlowIgDhigh stages, peritoneal CD5+ B cells were present, and serum Ig levels and in vivo responses to TI-II antigens were in the normal ranges. A comparable rescue by transgenic Btk expression was also observed in heterozygous Btk+/- female mice in those B-lineage cells that were Btk-deficient as a result of X chromosome inactivation. These findings indicate that the Btk- phenotype in the mouse can be corrected by expression of human Btk from the pre-B cell stage onwards.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed1997

The X-linked immunodeficiency defect in the mouse is corrected by expression of human Bruton's tyrosine kinase from a yeast artificial chromosome transgene.

Mutations in the gene for Bruton's tyrosine kinase result in the B cell differentiation defects X-linked agammaglobulinemia in man and X-linked immunodeficiency in mice. Here we describe the generation of two yeast artificial chromosome (YAC)-transgenic mouse strains in which high-level expression of human Btk is provided by endogenous regulatory cis-acting elements that are present on a 340-kb transgene, Yc340-hBtk. The expression pattern of the transgenic human Btk was found to parallel that of the endogenous murine gene. When the Yc340-hBtk-transgenic mice were mated onto a Btk-deficient background, the xid B cell defects were fully corrected: conventional and CD5+ B-1 B cells were present in normal numbers, serum IgM and IgG3 levels as well as responses to T cell-independent type II antigens were in the normal ranges. In vivo competition experiments in Btk+/- female mice demonstrated that in the conventional B cell population the Yc340-hBtk transgene could fully compensate the absence of expression of endogenous murine Btk. We conclude that in the YAC-transgenic mice Btk is appropriately expressed in the context of native regulatory sequences.

Agammaglobulinaemia Tyrosine Kinase↗

PubMed1997

CLIP-115, a novel brain-specific cytoplasmic linker protein, mediates the localization of dendritic lamellar bodies.

Intracellular localization of organelles may depend in part on specific cytoplasmic linker proteins (CLIPs) that link membranous organelles to microtubules. Here, we characterize rat cDNAs encoding a novel, brain-specific CLIP of 115 kDa. This protein contains two N-terminal microtubule-binding domains and a long coiled-coil region; it binds to microtubules and is homologous to CLIP-170, a protein mediating the binding of endosomes to microtubules. CLIP-115 is enriched in the dendritic lamellar body (DLB), a recently discovered organelle predominantly present in bulbous dendritic appendages of neurons linked by dendrodendritic gap junctions. Local microtubule depolymerization leads to a temporary reduction of DLBs. These results suggest that CLIP-115 operates in the control of brain-specific organelle translocations.

Amino Acid Sequence↗

PubMed1997

Regulation of the CRABP-I gene during mouse embryogenesis.

The cellular retinoic acid binding protein type I (CRABP-I) shows a highly specific expression pattern during mouse embryonic development. The tissues that express CRABP-I, i.e. the central nervous system (CNS), neural crest, branchial arches, limb bud and frontonasal mass, coincide with those that are most sensitive to unphysiological retinoic acid (RA) concentrations. We have investigated the transcriptional elements that are responsible for the spatiotemporal regulation of CRABP-I expression in the mouse embryo. We show here that a 16 kb fragment harbours all the elements needed for the correct spatiotemporal expression pattern. Upon further dissection of this fragment we have found that expression in the CNS is driven by elements in the upstream region of the gene, while expression in mesenchymal and neural crest tissue is regulated via element(s) located downstream of exon II of the gene. Two distinct fragments in the upstream region are required for expression in the CNS, as neither of these fragments alone is able to drive correct expression of a reporter gene in transgenic mice. DNAseI footprinting analysis of the two upstream fragments revealed the presence of a number of protected elements. One of these regulatory elements has the hallmarks of an RA response element, suggesting that CRABP-I expression in neural tissue can be directly modulated by RA via the RARs/RXRs.

Animals↗

PubMed1997

The effect of distance on long-range chromatin interactions.

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.

Animals↗

PubMed1997

The expression of bacterial nitroreductase in transgenic mice results in specific cell killing by the prodrug CB1954.

The enzyme nitroreductase, isolated from Escherichia coli B, converts CB1954 ((5-aziridin-1-yl)-2,4-dinitro-benzamide) into a cytotoxic DNA interstrand cross-linking agent. The E. coli B gene (nfnB, NTR) encoding nitroreductase (NTR) was cloned into eukaryotic expression vectors. When driven by a CMV promoter, 5-10% of the stably transfected mouse fibroblasts expressed the NTR enzyme. These cells were killed at a concentration of 20 microM CB1954 in comparison to nonexpressing cells which were killed at a much higher concentration (500 microM). We subsequently generated transgenic mice to test the prodrug system in vivo. Nitroreductase was expressed specifically in T cells driven by the control elements of the human CD2 locus. Upon CB1954 treatment, transgenic mice show extensive cell depletion in thymus and spleen (14-16% of normal cell numbers), whereas all other tissues are unaffected by prodrug administration. These results raise the possibility of using the NTR gene in anticancer therapy.

Animals↗

PubMed1997

The level of the tissue-specific factor GATA-1 affects the cell-cycle machinery.

GATA-1 is a tissue-specific DNA-binding protein containing two zinc-finger-like domains. It is expressed predominantly in erythrocytes. Consensus binding sites for GATA-1 have been found in the regulatory elements of all erythroid-specific genes examined. GATA-1 protein is required for erythroid differentiation beyond the proerythroblast stage. In this paper, we demonstrate that the overexpression of GATA-1 in murine erythroleukaemia (MEL) cells alleviates DMSO-induced terminal erythroid differentiation. Hence, there is no induction of globin gene transcription and the cells do not arrest in the G1 phase of the cell cycle. Furthermore, we demonstrate that expression of GATA-1 in non-transformed erythroid precursors also affects their proliferative capacity and terminal differentiation, as assayed by adult globin gene transcription. To gain insight into the mechanism of this effect, we studied the levels and activities of regulators of cell-cycle progression during DMSO-induced differentiation. A decrease in cyclin D-dependent kinase activity was observed during the induction of both control and GATA-1-overexpressing MEL cells. However, cyclin E-dependent kinase activity decreased more than 20-fold in control but less than 2-fold in GATA-1-overexpressing MEL cells upon induction. Thus GATA-1 may exert its effects by regulating cyclin E-dependent kinase activity. We also show that GATA-1 binds to the retinoblastoma protein in vitro, but not to the related protein p107, which may indicate that GATA-1 interacts directly with specific members of the cell-cycle machinery in vivo. We conclude that GATA-1 regulates cell fate, in terms of differentiation or proliferation, by affecting the cell-cycle apparatus.

Animals↗

PubMed1997

Gain adaptation and phase dynamics of compensatory eye movements in mice.

The vestibulocerebellum is involved in the control of compensatory eye movements. To investigate its role in the learning and timing of motor behaviour, eye movements in normal and mutant mice were investigated for the first time with the use of search coils. Wild-type mice showed the ability to increase the gain of their vestibulo-ocular reflex by visuo-vestibular training. This adaptation did not occur in lurcher mice, a natural mouse mutant that completely lacks Purkinje cells. During the optokinetic reflex the phase (timing) of the eye movements of lurchers lagged behind that of wild-type littermates, whereas during the vestibulo-ocular reflex it led that of the wild types. Ablations of different parts of the vestibulocerebellum indicated that the flocculus is necessary for the adaptation and the phase-leading effects of the cerebellum, whereas the nodulus might contribute to its phase-lagging effects. We conclude that Purkinje cells in the vestibulocerebellum are necessary for both learning and timing of compensatory eye movements in mice, and that the flocculus and nodulus may play antagonistic roles in these processes. The present description of the basic principles of cerebellar eye-movement control opens up the possibility to investigate the mechanisms of this motor behaviour at the molecular level in genetically manipulated mutant mice.

Adaptation, Physiological↗

PubMed1997

The murine homologue of HIRA, a DiGeorge syndrome candidate gene, is expressed in embryonic structures affected in human CATCH22 patients.

A wide spectrum of birth defects is caused by deletions of the DiGeorge syndrome chromosomal region at 22q11. Characteristic features include cranio-facial, cardiac and thymic malformations, which are thought to arise form disturbances in the interactions between hindbrain neural crest cells and the endoderm of the pharyngeal pouches. Several genes have been identified in the shortest region of deletion overlap at 22q11, but nothing is known about the expression of these genes in mammalian embryos. We report here the isolation of several murine embryonic cDNAs of the DiGeorge syndrome candidate gene HIRA. We identified several alternatively spliced transcripts. Sequence analysis reveals that Hira bears homology to the p60 subunit of the human Chromatin Assembly Factor I and yeast hir1p and Hir2p, suggesting that Hira might have some role in chromatin assembly and/or histone regulation. Whole mount in situ hybridization of mouse embryos at various stages of development show that Hira is ubiquitously expressed. However, higher levels of transcripts are detected in the cranial neural folds, frontonasal mass, first two pharyngeal arches, circumpharyngeal neural crest and the limb buds. Since many of the structures affected in DiGeorge syndrome derive from these Hira expressing cell populations we propose that haploinsufficiency of HIRA contributes to at least some of the features of the DiGeorge phenotype.

Alternative Splicing↗

PubMed1997

GATA-2 and GATA-3 regulate trophoblast-specific gene expression in vivo.

We previously demonstrated that the zinc finger transcription factors GATA-2 and GATA-3 are expressed in trophoblast giant cells and that they regulate transcription from the mouse placental lactogen I gene promoter in a transfected trophoblast cell line. We present evidence here that both of these factors regulate transcription of the placental lactogen I gene, as well as the related proliferin gene, in trophoblast giant cells in vivo. Placentas lacking GATA-3 accumulate placental lactogen I and proliferin mRNAs to a level 50% below that reached in the wild-type placenta. Mutation of the GATA-2 gene had a similar effect on placental lactogen I expression, but led to a markedly greater reduction (5- to 6-fold) in proliferin gene expression. Placentas lacking GATA-2 secrete significantly less angiogenic activity than wild-type placentas as measured in an endothelial cell migration assay, consistent with a reduction in expression of the angiogenic hormone proliferin. Furthermore, within the same uterus the decidual tissue adjacent to mutant placentas displays markedly reduced neovascularization compared to the decidual tissue next to wild-type placentas. These results indicate that GATA-2 and GATA-3 are important in vivo regulators of trophoblast-specific gene expression and placental function, and reveal a difference in the effect of these two factors in regulating the synthesis of related placental hormones.

Animals↗

PubMed1996

The role of EKLF in human beta-globin gene competition.

We have investigated the role of erythroid Kruppel-like factor (EKLF) in expression of the human beta-globin genes in compound EKLF knockout/human beta-locus transgenic mice. EKLF affects only the adult mouse beta-globin genes in homozygous knockout mice; heterozygous mice are unaffected. Here we show that EKLF knockout mice express the human epsilon and gamma-globin genes normally in embryonic red cells. However, fetal liver erythropoiesis, which is marked by a period of gamma- and beta-gene competition in which the genes are alternately transcribed, exhibits an altered ratio of gamma- to beta-gene transcription. EKLF heterozygous fetal livers display a decrease in the number of transcriptionally active beta genes with a reciprocal increase in the number of transcriptionally active gamma genes. beta-Gene transcription is absent in homozygous knockout fetuses with coincident changes in chromatin structure at the beta promoter. There is a further increase in the number of transcriptionally active gamma genes and accompanying gamma gene promoter chromatin alterations. These results indicate that EKLF plays a major role in gamma- and beta-gene competition and suggest that EKLF is important in stabilizing the interaction between the Locus Control Region and the beta-globin gene. In addition, these findings provide further evidence that developmental modulation of globin gene expression within individual cells is accomplished by altering the frequency and/or duration of transcriptional periods of a gene rather than changing the rate of transcription.

Animals↗

PubMed1996

Heterochromatin effects on the frequency and duration of LCR-mediated gene transcription.

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.

Animals↗

PubMed1996

Inactivation of Btk by insertion of lacZ reveals defects in B cell development only past the pre-B cell stage.

Bruton's tyrosine kinase (Btk) is a cytoplasmic protein kinase that is defective in X-linked agammaglobulinaemia in man and in X-linked immunodeficiency in the mouse. There is controversy regarding the stages of B cell development that are dependent on Btk function. To determine the point in B cell differentiation at which defects in Btk become apparent, we generated a mouse model by inactivating the Btk gene through an in-frame insertion of a lacZ reporter by homologous recombination in embryonic stem cells. The phenomenon of X-chromosome inactivation in Btk+/- heterozygous female mice enabled us to evaluate the competition between B cell progenitors expressing wild-type Btk and those expressing the Btk-/lacZ allele in each successive step of development. Although Btk was already expressed in pro-B cells, the first selective disadvantage only became apparent at the transition from small pre-B cells to immature B cells in the bone marrow. A second differentiation arrest was found during the maturation from IgD(low)IgM(high) to IgD(high)IgM(low) stages in the periphery. Our results show that Btk expression is essential at two distinct differentiation steps, both past the pre-B cell stage.

Agammaglobulinaemia Tyrosine Kinase↗