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Biomedical subjects

T Enver

Publications and source records attributed to T Enver.

At least 37 records · Page 2Linked to original sources
PubMed1994

Analysis of the developmental and transcriptional potentiation functions of 5'HS2 of the murine beta-globin locus control region in transgenic mice.

We analyze the role of 5'HS2 of the mouse beta-globin LCR in the transcriptional and developmental regulation of beta-globin gene expression. Previous studies have shown that the human beta-globin gene behaves as an adult gene in transgenic mice, being expressed in fetal liver and bone marrow-derived erythroblasts but not in yolk sac-derived embryonic erythroid cells. We show that linkage of mLCR5'HS2 to a human beta-globin gene alters this pattern of expression during ontogeny, resulting in expression of the linked beta-globin gene at all stages of murine erythroid development. Expression was independent of integration position and correlated with transgene copy number. Our results provide the first test of a phylogenetically homologous LCR in transgenic mice and demonstrate evolutionary conservation of both developmental and transcriptional potentiation functions between mammalian beta-globin LCRs.

Animals↗
PubMed1993

HEX: a novel homeobox gene expressed during haematopoiesis and conserved between mouse and human.

We describe the cloning of a novel homeodomain-containing gene, which is highly conserved between mouse and human. The human cDNA was initially isolated from human haematopoietic tissue and denoted HEX (haematopoietically expressed homeobox). Sequence analysis of the coding sequences from mouse and the partial cDNA from human shows that the homeodomain is most closely related to those of the HIx and HOX11 proteins. The HEX gene is present as a single copy in the human genome. Analysis of murine genomic DNA shows, in addition to an intron-containing gene homologous to HEX, the presence of a processed copy of the gene which has arisen within the last few million years. Analysis of human and murine haematopoietic cells and cell lines, revealed expression of the HEX gene in multipotential progenitors, as well as cells of the B-lymphocyte and myeloid lineages. However HEX was not expressed in T-lymphocytes or erythroid cells. This pattern of HEX gene expression suggests that it may play a role in haematopoietic differentiation.

Amino Acid Sequence↗
PubMed1993

Multiple changes in chromatin structure precede the transcriptional activation of the human growth hormone locus in placental cells.

In addition to the growth hormone gene (hGH-N) itself, the human growth hormone (hGH) locus contains four related genes, namely hGH-V and hCS-L, -A and -B, which have appeared very recently in evolution and are specifically expressed in placenta. With the aim of identifying the regulatory elements responsible for this placental-specific expression, we have mapped the DNaseI hypersensitive sites present at the hGH gene cluster in a placental cell line (BeWo) that expresses the hGH-V and hCS genes. Our results reveal a complex pattern of hypersensitive sites distributed along the hGH locus, most of which appear to be cell type-specific. Thus, we have identified placental-specific hypersensitive sites within the first intron of the hGH-N and hGH-V genes, but not in the equivalent regions of the hCS genes. In addition, we have found several placental-specific hypersensitive sites downstream of the hCS-L and hCS-A genes, which might reflect the presence of enhancer elements similar to that located downstream of the hCS-B gene (Walker et al. (1990) J. Biol. Chem. 265, 12940). Comparison of BeWo cells with a placental cell line (JEG-3) which does not express the hGH-V and hCS genes revealed a very similar pattern of hypersensitive sites, suggesting that the sites detected are established before the onset of transcription. Our results indicate that the transition to an active hGH locus in placental cells requires multiple alterations in chromatin structure, and provide a framework for the molecular analysis of the regulatory elements and mechanisms mediating such processes.

Biological Evolution↗
PubMed1992

Activation of the beta-globin locus control region precedes commitment to the erythroid lineage.

The beta-globin locus control region (LCR) is characterized by erythroid-specific DNase I hypersensitive sites and is involved in the chromatin organization, transcriptional potentiation, developmental regulation, and replication timing of the entire beta-globin gene cluster. When and how the LCR is first activated during erythropoiesis is not known. Here we analyze the chromatin structure of the LCR during early hematopoietic differentiation using nontransformed, multipotential, growth factor-dependent, murine hematopoietic progenitor cells. We show that LCR hypersensitive sites characteristic of erythroid cells are present in three independent multilineage progenitors [FDCP (factor-dependent cell, Paterson)-mix A4, B6SUtA, and LyD9] under conditions of self-renewal. Induction of differentiation down a nonerythroid pathway causes a progressive loss of hypersensitivity in the LCR. These results show that the beta-globin LCR is in an active chromatin configuration prior to erythroid commitment and indicate a significant role for selective gene repression in lineage specification.

Animals↗
PubMed1992

Cell lines produce factors that induce fetal hemoglobin in human BFUe-derived colonies.

Established cell lines were screened for secretion of activities than can stimulate fetal hemoglobin (HbF) production in adult burst-forming unit-erythroid (BFUe) cultures. Conditioned media from four cell lines, a human teratocarcinoma, an osteosarcoma, a bladder cell carcinoma, and feline leukemia virus (FeLV) A-infected feline fibroblasts (FEF-A cells), consistently increased the relative production of fetal globin in BFUe-derived colonies. In vitro translation of RNA from these cells in Xenopus oocytes yielded products that increased the gamma to gamma+beta ratio in adult erythroid colonies. These results demonstrate that a variety of cell lines produce factors that stimulate the production of HbF in vitro. The genes of such factors could be isolated by expression cloning of cDNA from cell lines using the Xenopus oocyte system.

Animals↗
PubMed1992

The mouse beta-globin locus control region: hypersensitive sites 3 and 4.

The human beta-globin LCR plays a key role in the transcriptional regulation of the beta-globin locus and comprises four erythroid specific DNase I hypersensitive sites, designated 5'HS1-4. We have now isolated genomic clones containing 5'HS3 and 5'HS4 of the mouse beta-globin LCR. 5'HS3 and 5'HS4 are located 15 kb and 22 kb upstream of the mouse epsilon y-globin gene, respectively. Sequence analysis of murine 5'HS3 and 5'HS4 reveals a significant degree of sequence conservation with their human homologues, including the presence of recognition sites for functionally relevant transcription factors. 5'HS3 and 5'HS4 regions were found to form hypersensitive sites in nuclei from murine erythroid cells, but not in nuclei from a variety of nonerythroid haematopoietic cell lines. Analysis of different mouse strains revealed the existence of a polymorphism that alters the spacing between 5'HS3 and 5'HS4. Taken together, our results emphasize the extent of evolutionary conservation and complexity of mammalian beta-globin LCRs. Finally, the cloning of mouse 5'HS3 and 5'HS4 will facilitate the molecular analysis of LCR function in the mouse model.

Animals↗
PubMed1992

Demonstration of a human epsilon-globin gene silencer with studies in transgenic mice.

The human epsilon-globin gene displays normal developmental regulation in transgenic mice; it is expressed only in embryonic and in definitive erythroid cells. We show here that deletion of a negative element located between -182 and -467 bp upstream of the epsilon-globin gene cap site results in continuation of epsilon gene expression in the definitive erythroblasts of the fetal liver and in the red blood cells of adult transgenic mice. These data provide direct in vivo evidence that cis acting silencing elements are involved in the developmental control of the epsilon-globin gene.

Animals↗
PubMed1991

Locus control region-A gamma transgenic mice: a new model for studying the induction of fetal hemoglobin in the adult.

All pharmacologic agents that induce fetal hemoglobin (Hb) have been discovered with in vivo studies of humans, macaques, and baboons. We tested whether transgenic mice carrying human fetal (gamma) globin genes provide a model for studying the pharmacologic induction of HbF in the adult. In initial studies, phenylhydrazine-induced hemolytic anemia, 5-azacytidine, butyrate, or combinations of these treatments failed to activate the human gamma-globin gene in a transgenic mouse line carrying a 4.4-kb G gamma globin gene construct that is expressed only in the embryonic stage of mouse development. Subsequently, adult mice carrying the human A gamma gene linked to the locus control region (LCR) regulatory sequences and expressing heterocellularly HbF (about 25%, gamma-positive cells) were used. Treatments with erythropoietin, 5-azacytidine, hydroxyurea, or butyrate resulted in induction of gamma gene expression as documented by measurement of F-reticulocytes, the gamma/gamma + beta biosynthetic ratio and the level of steady state gamma mRNA. Administration of erythropoietin or butyrate to transgenic mice carrying a muLCR-beta (human) globin construct, failed to increase human beta-globin expression. These results suggest that the muLCR-A gamma transgenic mice provide a new model for studying the induction of fetal Hb in the adult.

Animals↗
PubMed1991

Developmental regulation of fetal to adult globin gene switching in human fetal erythroid x mouse erythroleukemia cell hybrids.

Human fetal erythroid x murine erythroleukemia cell hybrids undergo human fetal (gamma) to adult (beta) globin gene switching in vitro under the control of a mechanism located on human chromosome 11. We investigated whether this mechanism acts in cis or in trans by preparing hybrid cells containing marked fragments of the gamma and beta genes known to switch in transgenic mice. In these cells the chromosomally introduced human globin locus undergoes the fetal to adult globin gene switch. In contrast, the marked globin gene fragments were expressed at all stages of hybrid development. These results suggest that either the mechanism of switching acts in cis or that sequences present in the chromosomal globin locus but missing from the transfected globin gene fragments mediate its action.

Animals↗
PubMed1991

Primary structure of the goat beta-globin locus control region.

The goat beta-globin cluster is composed of a triplicated four-gene set. A locus control region (LCR) containing elements homologous to 5'DNase I hypersensitive sites (HS) 1, 2, and 3 of the human beta-globin LCR has been identified at the 5' end of this locus. We determined 10.2 kb of nucleotide sequence from the goat beta-globin locus control region. Self-comparison of this sequence by dot matrix analysis revealed the presence of six complete and three incomplete artiodactyl repeats. A novel repeated element, termed D repeat, was also identified. Southern blotting analysis demonstrated that these elements exist in the goat genome as a low to medium frequency interspersed repeat family. The absence of any other large region of self-homology (direct or inverted) in the goat LCR suggests that 5'HSs 1, 2, and 3 did not arise through duplication, but rather evolved independently. By comparing goat 5'HS 1 to those of human, rabbit, and mouse, we show a greater than 80% conservation in sequence between the four species. This level of evolutionary conservation suggests that 5'HS 1 plays an important role in the regulation of beta-globin loci.

Animals↗
PubMed1991

Globin gene switching: a paradigm or what?

The delineation of the beta-globin locus control region has led to a new understanding of the developmental regulation of the beta-globin gene cluster. It now seems that globin gene switching is effected through the sequential and mutually exclusive interaction of the locus control region with the embryonic, fetal and adult stage specific globin genes.

Animals↗
PubMed1990

Autonomous developmental control of human embryonic globin gene switching in transgenic mice.

The mechanisms by which expression of the beta-like globin genes are developmentally regulated are under intense investigation. The temporal control of human embryonic (epsilon) globin expression was analyzed. A 3.7-kilobase (kb) fragment that contained the entire human epsilon-globin gene was linked to a 2.5-kb cassette of the locus control region (LCR), and the developmental time of expression of this construct was studied in transgenic mice. The human epsilon-globin transgene was expressed in yolk sac-derived primitive erythroid cells, but not in fetal liver or bone marrow-derived definitive erythroid cells. The absence of epsilon gene expression in definitive erythroid cells suggests that the developmental regulation of the epsilon-globin gene depends only on the presence of the LCR and the epsilon-globin gene itself (that is, an autonomous negative control mechanism). The autonomy of epsilon-globin gene developmental control distinguishes it from the competitive mechanism of regulation of gamma and beta-globin genes, and therefore, suggests that at least two distinct mechanisms function in human hemoglobin switching.

Animals↗
PubMed1990

Developmental regulation of human fetal-to-adult globin gene switching in transgenic mice.

Transgenic mice containing a human fetal (gamma-) or adult (beta-) globin gene linked to the beta-globin gene locus activation region (LAR) express the gene throughout development. By contrast, transgenic mice containing LAR linked to both a fetal and an adult globin gene display the normal developmental switch from fetal to adult gene expression. This suggests that the human fetal-to-adult globin gene switch is controlled through a mutually exclusive interaction between LAR and either the gamma- or beta-globin gene, resulting in the expression of only one gene at any given moment.

Animals↗
PubMed1990

Butyrate induces expression of transfected human fetal and endogenous mouse embryonic globin genes in GM 979 erythroleukemia cells.

We have analyzed the expression of endogenous murine genes and of transfected human fetal A gamma globin gene in GM 979, a mouse erythroleukemia line which produces adult as well as embryonic globins. Optimal induction of the endogenous murine adult globin genes was obtained with DMSO or HMBA while the epsilon y and beta h1 embryonic genes were preferentially induced by butyrate. Similarly, the transferred human A gamma-globin gene was preferentially induced by butyrate. These results as well as previous observations in vivo or in erythroid cell cultures suggest that butyrate preferentially induces the expression of fetal globin genes.

Acetamides↗
PubMed1990

Beta-globin locus activation regions: conservation of organization, structure, and function.

The human beta-globin locus activation region (LAR) comprises four erythroid-specific DNase I hypersensitive sites (I-IV) thought to be largely responsible for activating the beta-globin domain and facilitating high-level erythroid-specific globin gene expression. We identified the goat beta-globin LAR, determined 10.2 kilobases of its sequence, and demonstrated its function in transgenic mice. The human and goat LARs share 6.5 kilobases of homologous sequences that are as highly conserved as the epsilon-globin gene promoters. Furthermore, the overall spatial organization of the two LARs has been conserved. These results suggest that the functionally relevant regions of the LAR are large and that in addition to their primary structure, the spatial relationship of the conserved elements is important for LAR function.

Animals↗
PubMed1990

A deletion of the human beta-globin locus activation region causes a major alteration in chromatin structure and replication across the entire beta-globin locus.

Naturally occurring deletions that remove sequences located approximately 60 kb upstream of the human adult beta-globin gene result in the failure to transcriptionally activate the cis-linked globin genes in erythroid cells. In addition, transfection, transgenic, and somatic cell hybrid studies have revealed that sequences within this region are essential for the developmentally regulated high-level expression of cis-linked globin genes. This regulatory region located at the 5' end of the beta-globin locus has been termed the locus activation region (LAR). Using somatic cell hybrids, we have studied the chromatin structure and timing of DNA replication of the normal human beta-globin locus and a locus containing a de novo 25-kb deletion that removes elements of the LAR. As a result of this deletion, the entire beta-globin locus and sequences approximately 100 kb 5' and 3' of the adult beta-globin gene are DNase I-resistant and do not form characteristic distant hypersensitive sites. These sequences also replicate late in S phase in an erythroid cell background. In contrast, the sequences of the normal locus are DNase I sensitive and early replicating. These results suggest that the LAR is required for both the erythroid-specific chromatin structure and timing of DNA replication over a large physical distance.

Chromatin↗