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F Grosveld

Publications and source records attributed to F Grosveld.

At least 109 records · Page 6Linked to original sources
PubMed1994

The chicken beta/epsilon-globin enhancer directs autonomously regulated, high-level expression of the chicken epsilon-globin gene in transgenic mice.

In transiently transfected chicken erythroid cells, beta-like globin gene switching is mediated through differential activation of the cis-linked embryonic epsilon- and adult beta-globin genes by a shared enhancer. Two underlying mechanisms have been proposed: (i) tissue- and stage-specific factors activate the beta-globin promoter in adult erythroid cells (autonomous regulation); and (ii) the epsilon-globin promoter, although transcriptionally competent in both embryonic and adult cells, is suppressed at the adult stage through competition with the beta-globin promoter for interaction with the enhancer (competitive regulation). Analyses of transgenic mice carrying the chicken beta/epsilon-globin locus demonstrated that both genes depended on the enhancer for erythroid expression, but only the epsilon-globin gene exhibited developmentally appropriate transcription at levels comparable to the endogenous mouse globin genes. Surprisingly, the chicken epsilon-globin gene also appeared to be autonomously regulated, as has been observed for human embryonic and fetal beta-like globin genes in transgenic mice. These results suggest that the chicken beta/epsilon-globin enhancer possesses either embryonic stage or epsilon-globin gene specificity when incorporated into the murine germ line.

Animals↗
PubMed1994

Development of hematopoietic stem cell activity in the mouse embryo.

The precise time of appearance of the first hematopoietic stem cell activity in the developing mouse embryo is unknown. Recently the aorta-gonad-mesonephros region of the developing mouse embryo has been shown to possess hematopoietic colony-forming activity (CFU-S) in irradiated recipient mice. To determine whether the mouse embryo possesses definitive hematopoietic stem cell activity in the analogous AGM region and to determine the order of appearance of stem cells in the yolk sac, AGM region, and liver, we transferred these embryonic tissues into adult irradiated recipients. We report here the long-term, complete, and functional hematopoietic repopulation of primary and serial recipients with AGM-derived cells. We observe potent hematopoietic stem cell activity in the AGM region before the appearance of yolk sac and liver stem cell activity and discuss a model for the maturation of stem cell activity in mouse embryogenesis.

Animals↗
PubMed1994

Chromatin domains as potential units of eukaryotic gene function.

Many current models for eukaryotic gene activation and regulation postulate that higher order chromatin structures act as major modulators of gene function. Genetic evidence suggests that nucleosomes and more specifically targeted proteins, such as Polycomb in Drosophila and SIR3 in Saccharomyces cerevisiae, are involved in creating repressive chromatin structures. In addition, the discovery of locus control regions in vertebrates suggests that the primary information for gene activation can reside entirely in specific combinations of transcription factor binding sites. Difficulties associated with experimental design and interpretation make the investigation of whether domains have discrete functional boundaries problematic, and the concept of the chromatin domain as an integrated structural and functional unit remains to be established.

Animals↗
PubMed1994

Embryonic expression and cloning of the murine GATA-3 gene.

We describe the embryonic expression pattern as well as the cloning and initial transcriptional regulatory analysis of the murine (m) GATA-3 gene. In situ hybridization shows that mGATA-3 mRNA accumulation is temporally and spatially regulated during early development: although found most abundantly in the placenta prior to 10 days of embryogenesis, mGATA-3 expression becomes restricted to specific cells within the embryonic central nervous system (in the mesencephalon, diencephalon, pons and inner ear) later in gestation. GATA-3 also shows a restricted expression pattern in the peripheral nervous system, including terminally differentiating cells in the cranial and sympathetic ganglia. In addition to this distinct pattern in the nervous system, mGATA-3 is also expressed in the embryonic kidney and the thymic rudiment, and further analysis showed that it is expressed throughout T lymphocyte differentiation. To begin to investigate how this complex gene expression pattern is elicited, cloning and transcriptional regulatory analyses of the mGATA-3 gene were initiated. At least two regulatory elements (one positive and one negative) appear to be required for appropriate tissue-restricted regulation after transfection of mGATA-3-directed reporter genes into cells that naturally express GATA-3 (T lymphocytes and neuroblastoma cells). Furthermore, this same region of the locus confers developmentally appropriate expression in transgenic mice, but only in a subset of the tissues that naturally express the gene.

Animals↗
PubMed1994

The mode of anchorage to the cell surface determines both the function and the membrane location of Thy-1 glycoprotein.

The surface glycoprotein, Thy-1, when expressed by transfection in NG115/401L neural cells, inhibits their neurite outgrowth over astrocytes. We have investigated the role of the glycosylphosphatidylinositol anchor of Thy-1 in this inhibition. Hybrid molecules, in which the lipid anchor was replaced by polypeptide transmembrane domains, were expressed by transfection. Lines expressing Thy-1 with the transmembrane and full cytoplasmic domains of NCAM-140, or with the transmembrane and truncated cytoplasmic domain of CD8, were not inhibited in their ability to extend neurites over astrocytes. Truncation of the cytoplasmic domain of NCAM-140 to just two amino acids, however, produced a transmembrane form of Thy-1 that, when expressed at high levels, inhibited neurite outgrowth. All forms of Thy-1 were concentrated in clusters that occurred primarily on fine filopodia. In double transfectants expressing normal Thy-1 and Thy-1 with the full NCAM cytoplasmic tail, the clusters of each form were separate, with no instances of the transmembrane form being found within the clusters of lipid-anchored Thy-1. Thy-1 with the two-amino-acid cytoplasmic domain of NCAM also occurred in clusters separate from those occupied by lipid-anchored Thy-1, but substantial 'invasion' of the clusters of normal Thy-1 by this transmembrane construct occurred. We suggest that the ability of this hybrid protein to enter the lipid-anchored clusters enables it to activate the signalling pathways that normal Thy-1 uses. Thus the membrane anchor, in targetting Thy-1 to different microdomains on the cell surface, determines its ability to inhibit neurite outgrowth on astrocytes.

Amino Acid Sequence↗
PubMed1993

The two zinc finger-like domains of GATA-1 have different DNA binding specificities.

The GATA-1 transcription factor has been shown to be important in the regulation of globin and non-globin genes in erythroid, megakaryocytic and mast cell lineages. It is a member of a family of GATA proteins which both overlap in their expression patterns and bind the motif (A/T)GATA(A/G). The GATA family of proteins are also members of the superfamily of zinc finger-like domain proteins and have two similar domains of the type Cys-X2-Cys-X17-Cys-X2-Cys which direct the DNA binding of the protein. A random oligonucleotide selection procedure has been employed to further elucidate the mechanism of GATA-1-DNA recognition. The resulting oligonucleotides were tested for binding activity to both wild-type and mutant GATA-1 proteins. Two classes of GATA-1-DNA interaction have been defined, the first requiring only the carboxy finger of GATA-1 to bind and having the motif GAT(A/T), the second requiring both finger domains to bind and having the core motif (T/C)AAG. By using sequence comparison and depurination analysis it is concluded that the two finger-like domains of GATA-1 have different DNA binding recognition motifs. Binding of GATA-1 to GAT(A/T) motifs is associated with transcriptional activation of linked genes. The only known (T/C)AAG motif is in the distal CAAT-box promoter region of the human A gamma-globin gene where the binding of GATA-1 appears to regulate the correct developmental suppression of gamma-globin expression.

Animals↗
PubMed1993

Estrogen-inducible and liver-specific expression of the chicken Very Low Density Apolipoprotein II gene locus in transgenic mice.

We have examined the chicken Very Low Density Apolipoprotein II (apoVLDL II) gene locus in transgenic mice. A DNA fragment composed of the transcribed region, 16 kb of 5' flanking and 400 bp of 3' flanking sequences contained all the information sufficient for estrogen-inducible, liver-specific expression of the apoVLDL II gene. The far-upstream region contains a Negative Regulating Element coinciding with a DNaseI-hypersensitive site at -11 kb. In transgenic mice, the NRE at -11 kb is used for downregulating the expression to a lower maximum level. The NRE might be used for modulating apoVLDL II gene expression, and may be involved in the rapid shut-down of the expression after hormone removal.

Animals↗
PubMed1993

The regulation of human globin gene switching.

This paper describes the mechanism of regulation of the human beta-globin on the basis of a number of natural mutations and experiments in transgenic mice. From these data we conclude that this multigene locus is regulated at a number of different levels involving specific interactions between the Locus Control Region (LCR) and the individual genes. Most important is the action of stage specific transcription factors acting on sequences immediately flanking the genes. In addition, specificity is obtained through specific interaction of the genes with the LCR and through competition of the genes for interaction with the LCR.

Animals↗
PubMed1993

Functional analysis of the human neurofilament light chain gene promoter.

We have carried out a structural and functional analysis on the human NF-L (H-NF-L) gene. It contains a methylation-free island, spanning the 5' flanking sequences and the first exon and a number of neuronal-specific DNase I hypersensitive sites have been identified in the upstream region as well as within the body of the gene. Analysis in cell lines and transgenic mice using a combination of these sites has revealed the presence of a conserved element(s) between -300bp and -190bp which is required for neuronal-specific expression.

Animals↗
PubMed1993

Synthetic human beta-globin 5'HS2 constructs function as locus control regions only in multicopy transgene concatamers.

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.

Animals↗
PubMed1993

The minimal requirements for activity in transgenic mice of hypersensitive site 3 of the beta globin locus control region.

Proper expression of the human beta-like globin genes is completely dependent on the presence of the locus control region or LCR, a region containing four DNase hypersensitive sites (HS1-4) situated 5' to the structural genes. Linkage of the LCR to a transgene results in copy number-dependent transcription, independent of the site of integration in the host genome. We have analysed a small region of the LCR (HS3) in transgenic animals to determine the minimal interactions that are required for this property. The results show that a specific combination of a G-rich sequence flanked on each side by one binding site for the transcription factor GATA1 is essential to obtain position-independent expression of a linked beta globin gene in erythroid cells. The overall transcriptional activity of HS3 is achieved through synergy with other combinations of similar binding sites.

Animals↗
PubMed1993

Transcriptional regulation of multigene loci: multilevel control.

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.

Animals↗
PubMed1993

The regulation of human globin gene expression.

The haemopoietic system provides a well-characterized and accessible system for studying the mechanisms of developmental regulation and differentiation in higher eukaryotes. Our current understanding of the steps involved in the early stages of differentiation are poorly understood but a great deal is now known about the mechanisms by which globin expression is regulated in cells committed to the erythroid lineage. Many of the critical cis-acting sequences and some of the important trans-acting factors involved have been identified and current work is focusing on how these interact to produce high levels of tissue-specific and developmentally regulated expression of the human globin genes.

Animals↗
PubMed1993

Each hypersensitive site of the human beta-globin locus control region confers a different developmental pattern of expression on the globin genes.

We have tested the effect of the individual DNase I hypersensitive site (HS) regions of the globin locus control region (LCR) on the developmental expression pattern of the human gamma and beta genes in transgenic mice. The results show that HS3 is the most active site during the embryonic period. It is also the only site capable of high level expression of the gamma genes during fetal hematopoiesis, in a population of cells that are capable of expressing both the gamma and beta genes. Region HS4 shows the highest activity during the adult stage and expresses the gamma genes only at low levels during the embryonic period. HS2 drives equivalent levels of gamma or beta transgene expression throughout development. HS1 has a similar pattern to HS2, although the activity of HS1 is very low. From these results we conclude that the HS regions have distinct developmental specificities and suggest that in the complete LCR they interact with each other to form a larger complex which, in turn, interacts with the globin genes.

Animals↗
PubMed1993

Regulated expression of K+ channel genes in electrically silent mammalian cells by linkage to beta-globin gene-activation elements.

Fundamental studies of the mechanism of human beta-like globin gene-expression have identified DNA sequences (locus control regions or LCRs) which directly influence the specific activation of beta-globin genes in erythroid cells. Here we report the first applications of LCR-mediated gene-activation to stable electrophysiological expression of several homomultimeric ion channel proteins. We describe expression driven from a native K+ channel gene promoter region, contiguous to an intronless coding sequence, within a single excised human genomic DNA fragment. In addition, cDNAs encoding both inactivating and non-inactivating mammalian K+ currents have been expressed by insertion between the promoter and second intron of the human beta-globin gene. Expression levels are characteristically independent of integration position and are proportional to LCR-gene copy number, a parameter specific for each clonal cell line. K+ channel expression is inducible by erythroid differentiation and has been demonstrated by electrophysiological recordings of cells taken directly from culture.

Amino Acid Sequence↗
PubMed1992

A single point mutation is the cause of the Greek form of hereditary persistence of fetal haemoglobin.

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.

Animals↗