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Biomedical subjects

J D Engel

Publications and source records attributed to J D Engel.

At least 91 records · Page 5Linked to original sources
PubMed1993

Human T cell transcription factor GATA-3 stimulates HIV-1 expression.

A family of transcriptional activating proteins, the GATA factors, has been shown to bind to a consensus motif through a highly conserved C4 zinc finger DNA binding domain. One member of this multigene family, GATA-3, is most abundantly expressed in T lymphocytes, a cellular target for human immunodeficiency virus type 1 (HIV-1) infection and replication. In vitro DNase I footprinting analysis revealed six hGATA-3 binding sites in the U3 region (the transcriptional regulatory domain) of the HIV-1 LTR. Cotransfection of an hGATA-3 expression plasmid with a reporter plasmid whose transcription is directed by the HIV-1 LTR resulted in 6- to 10-fold stimulation of LTR-mediated transcription, whereas site specific mutation of these GATA sites resulted in virtual abrogation of the activation by hGATA-3. Further, deletion of the hGATA-3 transcriptional activation domain abolished GATA-dependent HIV-1 trans-activation, showing that the stimulation of viral transcription observed is a direct effect of cotransfected hGATA-3. Introduction of the HIV-1 plasmids in which the GATA sites have been mutated into human T lymphocytes also caused a significant reduction in LTR-mediated transcription at both the basal level and in (PHA- plus PMA-) stimulated T cells. These observations suggest that in addition to its normal role in T lymphocyte gene regulation, hGATA-3 may also play a significant role in HIV-1 transcriptional activation.

Animals↗
PubMed1993

Erythroid transcription factor GATA-1 is abundantly transcribed in mouse testis.

The transcription factor GATA-1 is a fundamental regulator of genes in haematopoietic cell lineages and belongs to a family of factors that bind to the consensus sequence WGATAR. The GATA motif was originally identified in cis-regulatory regions of globin and other erythroid-specific genes, but the range of genes controlled by the GATA factors has since expanded. Members of the GATA transcription factor family share a conserved zinc-finger DNA-binding domain, but the expression profile of each GATA factor is distinct. Here we show that a testis form of murine (m)GATA-1 messenger RNA is transcribed from a promoter located 5' to the erythroid first exon, and the remaining exons (which encode the mGATA-1 protein) are used in common by both testis and erythroid transcripts. We use an anti-mGATA-1 monoclonal antibody to show that the factor expressed in erythroid cells is the same as that found in the seminiferous tubules of murine testis. The GATA-1-expressing cells in 10-week-old testis were found only in contact with the basement membrane of seminiferous tubules, suggesting that GATA-1 regulates genes during the earliest stages of spermatogenesis.

Aging↗
PubMed1993

Quantitation of RNA using the polymerase chain reaction.

Sequential use of reverse transcriptase and the polymerase chain reaction (RT-PCR) permits rapid and sensitive detection of specific RNAs. However, the greatest advantage of RT-PCR, its remarkable sensitivity, has also limited its usefulness in quantitative applications, since the effects of minor variations in reaction conditions from sample to sample are greatly magnified during the amplification process. Several recently developed techniques circumvent this problem, allowing accurate quantitation of RNA using RT-PCR.

Animals↗
PubMed1993

Developmental regulation of human beta-globin gene transcription: a switch of loyalties?

Synthesis of different hemoglobin polypeptides during the early stages of human development is principally regulated by transcriptional control mechanisms that determine which of the five beta-type globin genes is expressed. The means by which this is achieved have been scrutinized for several decades, and insights have been gained from introducing segments of the human beta-globin locus into transgenic mice, and from analysis of naturally occurring mutations at the locus. I describe here a model which attempts to resolve several of the current puzzles and provides simple, testable predictions for how differential beta-globin gene transcription might be achieved during human development.

Animals↗
PubMed1993

Ectopic expression of a conditional GATA-2/estrogen receptor chimera arrests erythroid differentiation in a hormone-dependent manner.

The GATA factors are a family of transcriptional regulatory proteins in eukaryotes that share extensive homology in their DNA-binding domains. One enigmatic aspect of GATA factor expression is that several GATA proteins, which ostensibly share the same DNA-binding site specificity, are coexpressed in erythroid cells. To elucidate the roles of individual GATA factors in erythropoiesis, conditional alleles of GATA-1, GATA-2, and GATA-3 were prepared by fusing each of the factors to the hormone-binding domain of the human estrogen receptor (ER). These GATA/ER chimeric factors were shown to be hormone-inducible trans-activating proteins in transient transfection assays. When stably introduced into primary erythroblasts or conditionally transformed erythroid progenitors cells, exogenous GATA-2/ER promoted proliferation and inhibited terminal differentiation in an estrogen-dependent manner. These phenotypic effects are specifically attributable to the action of ectopically expressed GATA-2/ER because erythroblasts expressing exogenous GATA-2 are constitutively arrested in differentiation and because erythroid progenitors expressing either Gal/ER or GATA-3/ER do not display a hormone-responsive block in differentiation. Thus, the GATA-2 transcription factor appears to play a role in regulating the self-renewal capacity of early erythroid progenitor cells.

Amino Acid Sequence↗
PubMed1993

DNA-binding specificities of the GATA transcription factor family.

Members of the GATA family of transcription factors, which are related by a high degree of amino acid sequence identity within their zinc finger DNA-binding domains, each show distinct but overlapping patterns of tissue-restricted expression. Although GATA-1, -2, and -3 have been shown to recognize a consensus sequence derived from regulatory elements in erythroid cell-specific genes, WGATAR (in which W indicates A/T and R indicates A/G), the potential for more subtle differences in the binding preferences of each factor has not been previously addressed. By employing a binding selection and polymerase chain reaction amplification scheme with randomized oligonucleotides, we have determined the binding-site specificities of bacterially expressed chicken GATA-1, -2, and -3 transcription factors. Whereas all three GATA factors bind an AGATAA erythroid consensus motif with high affinity, a second, alternative consensus DNA sequence, AGATCTTA, is also recognized well by GATA-2 and GATA-3 but only poorly by GATA-1. These studies suggest that all three GATA factors are capable of mediating transcriptional effects via a common erythroid consensus DNA-binding motif. Furthermore, GATA-2 and GATA-3, because of their distinct expression patterns and broader DNA recognition properties, may be involved in additional regulatory processes beyond those of GATA-1. The definition of an alternative GATA-2-GATA-3 consensus sequence may facilitate the identification of new target genes in the further elucidation of the roles that these transcription factors play during development.

Base Sequence↗
PubMed1993

Expression of the chicken GATA factor family during early erythroid development and differentiation.

The DNA motif WGATAR has been identified within transcriptional regulatory domains of globin and other erythroid-specific genes and the activator proteins that bind to this regulatory element, the GATA factors, belong to a multi-gene family that is expressed in chicken erythroid cells. Here we show that, as in chickens, multiple members of the GATA factor family are expressed in human and murine erythroid cells. During the early stages of chicken embryogenesis (well before blood island formation), each of the GATA family members is transcribed with a unique temporal and spatial pattern. In the primitive erythroid lineage, transcription of the embryonic epsilon-globin gene parallels GATA-1 expression while the switch to beta-globin transcription in definitive erythroid cells is directly preceded by a pronounced increase in GATA-3 accumulation. The timing and pattern of expression of these different mRNAs during avian erythroid development and differentiation suggests that temporally regulated changes in GATA factor expression are required for vertebrate hematopoiesis.

Animals↗
PubMed1993

A GATA family transcription factor is expressed along the embryonic dorsoventral axis in Drosophila melanogaster.

The GATA transcription factors are a family of C4 zinc finger-motif DNA-binding proteins that play defined roles in hematopoiesis as well as presumptive roles in other tissues where they are expressed (e.g., testis, neuronal and placental trophoblast cells) during vertebrate development. To investigate the possibility that GATA proteins may also be involved in Drosophila development, we have isolated and characterized a gene (dGATAa) encoding a factor that is quite similar to mammalian GATA factors. The dGATAa protein sequence contains the two zinc finger DNA-binding domain of the GATA class but bears no additional sequence similarity to any of the vertebrate GATA factors. Analysis of dGATAa gene transcription during Drosophila development revealed that its mRNA is expressed at high levels during early embryogenesis, with transcripts first appearing in the dorsal portion of the embryo just after cellularization. As development progresses, dGATAa mRNA is present at high levels in the dorsal epidermis, suggesting that dGATAa may be involved in determining dorsal cell fate. The pattern of expression in a variety of dorsoventral polarity mutants indicates that dGATAa lies downstream of the zygotic patterning genes decapentaplegic and zerknüllt.

Amino Acid Sequence↗
PubMed1992

Cloning and expression of chicken erythrocyte transglutaminase.

We report the sequences of cDNAs encoding chicken erythrocyte transglutaminase (EC 2.3.2.13). The complete mRNA consists of 3345/3349 nucleotides and predicts a single open reading frame. Nine peptide sequences derived from partial digests of the isolated protein agreed with the corresponding translation of the open reading frame. Approximately 60% identities between the avian protein and three related mammalian enzymes were found. Chicken erythrocyte transglutaminase mRNA is most abundant in red blood cells and kidney, and it accumulates during erythroid cell differentiation.

Amino Acid Sequence↗
PubMed1992

Developmental potential.

In summary (and probably to no one's genuine surprise), it seems clear that some of the key themes in the mechanisms employed during development reiterate themselves throughout the animal kingdom. Yet, as our understanding becomes more refined, new and beguiling observations point to unique aspects of each developmental program. The concentration and absolute position of a variety of positional signaling molecules is likely to be very important in determinative events (establishment of the anteroposterior positioning in a field as in retinal development, establishment or enactment of a hox code, and selector gene regulation through gradients in Drosophila). Appropriate signalling responses are virtually certain to depend critically on the appropriate expression of each component of cellular signal transduction pathways (initiated by the activation of cell-surface receptor protein kinases to finally eliciting gene expression changes through the differential activity of specific transcription factors). The important biochemical details of transcription factor activation of specific respondent genes may be either simpler (as indicated from the murine/Drosophila domain swap experiments) or more complicated (from the responses of mim-1 to cellular versus viral myb proteins) than we had heretofore anticipated.

Animals↗
PubMed1992

Individual stage selector element mutations lead to reciprocal changes in beta- vs. epsilon-globin gene transcription: genetic confirmation of promoter competition during globin gene switching.

Biochemical and genetic analysis of the embryonic to adult beta-like globin gene switch in chickens has led to the hypothesis that competition between the promoters of the cis-linked epsilon- and beta-globin genes for interaction with a shared enhancer mediates the developmental changes in expression of beta-globin protein isotypes. To test specific predictions of this promoter competition model, a sensitive RNA/polymerase chain reaction assay has been used to investigate the effects of individual beta-globin promoter mutations on expression of the two linked genes in transiently transfected erythroid cells. Mutations that attenuated adult beta-globin transcription resulted concomitantly in a proportional increase in expression of the embryonic epsilon-globin gene. Consistent with the model, mutations disrupting the binding sites for either of two adult stage-specific transcription factors (NF-E4 and beta CTF) indicate that these sites are essential both for induction of beta-globin gene expression and for indirect suppression (through promoter competition) of epsilon-globin transcription in definitive (adult) erythroid cells. These results provide direct evidence that stage-specific transcription factors affect the equilibrium existing between multiple interacting globin cis-regulatory elements. We conclude that promoter competition is an important mechanism through which developmental regulation of chicken beta-globin gene switching is achieved and that such competitive interactions may prove to be generally applicable to the regulation of a variety of other temporally or spatially restricted gene expression patterns.

Animals↗
PubMed1992

cis and trans regulation of tissue-specific transcription.

Analysis of both the cis-regulatory sequences which control globin gene switching as well as the trans-acting factors which bind to these sequences to elicit a differential, developmentally regulated response has lent insight into the general mechanisms responsible for tissue-specific gene regulation. We show here that the chicken adult beta-globin gene promoter sequences are intimately involved in competitive interaction with the beta/epsilon-globin enhancer to regulate differentially epsilon- versus beta-globin gene transcription. Secondly, we show that the family of GATA transcription factors directs gene regulation in a variety of discrete cell types, and describe potential cellular target genes for each member of the GATA factor family, as well as potential mechanisms whereby multiple GATA factors expressed in a single cell might be used to elicit differential transcriptional activities.

Animals↗
PubMed1991

Murine and human T-lymphocyte GATA-3 factors mediate transcription through a cis-regulatory element within the human T-cell receptor delta gene enhancer.

A family of transcriptional activators has recently been identified in chickens; these transcriptional activators recognize a common consensus motif (WGATAR) through a conserved C4 zinc finger DNA-binding domain. One of the members of this multigene family, cGATA-3, is most abundantly expressed in the T-lymphocyte cell lineage. Analysis of human and murine GATA-3 factors shows a striking degree of amino acid sequence identity and similar patterns of tissue specificity of expression in these three organisms. The murine and human factors are abundantly expressed in a variety of human and murine T-cell lines and can activate transcription through a tissue-specific GATA-binding site identified within the human T-cell receptor delta gene enhancer. We infer that the murine and human GATA-3 proteins play a central and highly conserved role in vertebrate T-cell-specific transcriptional regulation.

Amino Acid Sequence↗