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J D Engel

Publications and source records attributed to J D Engel.

At least 55 records · Page 3Linked to original sources
PubMed1998

A core region of the mafK gene IN promoter directs neurone-specific transcription in vivo.

BACKGROUND: MafK serves as a required subunit of erythroid transcription factor NF-E2 and also functions with various heterodimeric CNC family proteins. MafK expression begins in early mesoderm and is observed in mesenchymal and haematopoietic cells, as well as in neurones during mouse development. In mesodermal descendants, MafK mRNA begins with a distal first exon (called IM), whereas the mRNA in neurones begins with a proximal first exon (IN). RESULTS: To elucidate the mechanisms that underlie the tissue-specific transcription of the mafK gene, and to gain insights into the functions of MafK during neural development, we analysed the activity of the mafK IN promoter. A detailed investigation of mafK expression in the embryonic spinal cord revealed that IN-initiated mRNA is expressed in the ventral side of the spinal cord. Transient transfection analysis of reporter plasmids bearing the IN promoter and upstream regions revealed that the 'core' region of this promoter (nt -67 to -9) is active and that its integrity is crucial for this activity. The core region was also capable of directing the tissue-specific transcription of a reporter gene in neural cells of the spinal cord in transgenic mice in vivo. CONCLUSION: These results demonstrate that the specific expression of mafK in neural cells is determined, at least in part, by the core region of the IN promoter.

Animals↗
PubMed1998

Absence of expression of transforming growth factor-beta type II receptor is associated with an aggressive growth pattern in a murine renal carcinoma cell line, Renca.

Transforming growth factor-beta1 (TGF-beta1) inhibits the proliferation of many cancer cells. However, tumor cells frequently become resistant to this inhibitory effect due to the absence of TGF-beta receptor (TbetaR) expression. This study reports the nature of TGF-beta sensitivity in an aggressive murine renal carcinoma cell line, Renca, investigated in a series of experiments. The growth of Renca cells, in tissue culture, was not sensitive to the inhibitory effect of TGF-beta1 with doses ranging from 0.1 to 10 ng./ml., nor was this cell line sensitive to the effect of TGF-beta1 in inducing the expression of plasminogen activator inhibitor-I. Renca cells expressed TGF-beta1 mRNA and protein, as determined by RT-PCR and ELISA, respectively. The level of TGF-beta1 production by Renca cells was moderate, thus eliminating the possibility that endogenous TGF-beta1 production might be masking the effect of TGF-beta sensitivity. Furthermore, Renca cells expressed TbetaR-I mRNA, but did not express TbetaR-II mRNA, suggesting that the absence of this receptor may be the cause of TGF-beta insensitivity. Additionally, a vector containing the TbetaR-II cDNA was transiently transfected into Renca cells. The inhibitory effect of TGF-beta1 was introduced in Renca cells after transfection with this receptor. At the same time, the growth rate of these cells diminished significantly when compared with that of the wild type Renca cells, as judged by the rate of [3H]-thymidine incorporation in the absence of any exogenous TGF-beta1. These observations demonstrated that Renca cells lack the functional TbetaR-II and suggest that their aggressive growth pattern is due, at least in part, to their insensitivity to TGF-beta.

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

The world according to Maf.

Maf family proteins are so named because of their structural similarity to the founding member, the oncoprotein v-Maf. The small Maf proteins (MafF, MafG and MafK), as do all family members, include a characteristic basic region linked to a leucine zipper (b-Zip) domain which mediate DNA binding and subunit dimerization respectively. The small Maf proteins form homodimers or heterodimers with other b-Zip proteins present in the cell and bind to Maf recognition elements (MARE) in DNA. Since they lack known transcriptional activation domains, the small Maf proteins function either as obligatory heterodimeric partner molecules with numerous large subunits, discussed below, or alternatively as homo- or heterodimeric transcriptional repressors. The three small Maf proteins are expressed in a number of overlapping tissues, but their expression profiles nonetheless appear to be under meticulous tissue- and developmental stage-specific control. The MARE bears a striking resemblance to the NF-E2 binding sequence. NF-E2 binding sites in the human beta-globin locus control region have been directly implicated as integral components in the circuitry required for eliciting changes in chromatin structure that precede globin gene activation. While the NF-E2 DNA sequence has been shown to be important for erythroid-specific gene regulation, a growing list of other genes may also be regulated through the same, or very similar, cis elements in non-erythroid cells. Taken together, these observations argue that comprehensive analysis of the activities of the small Maf proteins may provide a unique perspective for expanding our understanding of transcriptional regulation that can be elicited through interacting transcription factor networks.

Animals↗
PubMed1997

GATA-1 transcription is controlled by distinct regulatory mechanisms during primitive and definitive erythropoiesis.

Transcription factor GATA-1 is required for the terminal differentiation of both the primitive and definitive erythroid cell lineages, and yet the regulatory mechanisms of GATA-1 itself are not well understood. To clarify how the GATA-1 gene is transcriptionally controlled in vivo, presumptive regulatory regions of the gene were tested by fusion to a reporter gene and then examined in transgenic mice. We found that a transcriptional control element located between -3.9 and -2.6 kb 5' to the erythroid first exon serves as an activating element and that this sequence alone is sufficient to recapitulate the expression of GATA-1 (but uniquely in primitive erythroid cells). Addition of sequences from the GATA-1 first intron to this upstream element provides a necessary and sufficient condition for complete recapitulation of GATA-1 expression in both primitive and definitive erythroid cells. The first intron element does not possess intrinsic transcriptional activation potential when linked to the GATA-1 gene promoter but rather requires the upstream activating element for its activity. These experiments show that GATA-1 gene expression is regulated by discrete transcriptional control elements during definitive and primitive erythropoiesis: The 5' element displays properties anticipated for a primitive erythroid cell-specific activating element, and the novel element within the GATA-1 first intron specifically augments this activity in definitive erythroid cells.

Animals↗
PubMed1997

Mutation of gene-proximal regulatory elements disrupts human epsilon-, gamma-, and beta-globin expression in yeast artificial chromosome transgenic mice.

Previous studies have defined transcriptional control elements, in addition to the promoters, that both lie near individual human beta-globin locus genes and have been implicated in their differential stage-specific regulation during development (i.e., are believed to directly participate in hemoglobin switching). We have reinvestigated the activities during erythropoiesis that might be conferred by two of the more intensively analyzed of these elements, the epsilon-globin gene 5' silencer and the beta-globin gene 3' enhancer, by deleting them from a yeast artificial chromosome that spans the human beta-globin locus, and then analyzing transgenic mice for expression of all of the human genes. These studies show that sequences within the epsilon-globin "silencer" are not only required for silencing but are also required for activation of epsilon-globin transcription; furthermore, deletion of the silencer simultaneously reduced gamma-globin transcription during the yolk sac stage of erythroid development. Analysis of the adult beta-globin gene 3' enhancer deletion showed that its deletion affects only that gene.

Animals↗
PubMed1997

Upstream and downstream of erythroid transcription factor GATA-1.

All mature blood lineages in the peripheral circulation are derived from pluripotent haematopoietic stem cell. Progressive lineage-restriction of this stem cell is executed, in part, by the interplay and cross-talk between a host of lineage-restricted as well as ubiquitous transcription factors. To elucidate the regulatory mechanisms underlying the erythroid gene regulation, it is essential to understand how individual transcription factors contribute to the regulation of specific target genes, and how these erythroid transcription factor genes are regulated in turn. These key issues of mammalian development have been addressed by examining the activities controlling the prototype transcription factor, GATA-1. The transcriptional regulation of GATA-1 has been intensively investigated, thereby leading to the identification of its developmental stage-specific regulatory sequences. Loss-of-function mutant animals, combined with specific marking of the primitive and definitive erythroid lineages have also shed new insight into how GATA-1 activity is required in vivo at specific developmental stages. Procedures have also been developed for ascertaining whether or not the GATA-1 protein actually binds in vivo to regulatory GATA motifs in candidate target genes. Application of a similar multifaceted approach should enable investigators to examine the physiological roles that any transcription factor might play in vivo during the differentiation of any well defined cell lineage.

Animals↗
PubMed1997

Conserved structure, regulatory elements, and transcriptional regulation from the GATA-1 gene testis promoter.

Transcription factor GATA-1 was first identified in erythroid cells, but was later shown to also be expressed in Sertoli cells of the mouse testis. GATA-1 transcription in testis initiates from a different first exon (exon IT) than the erythroid mRNA (transcribed from exon IE). To begin to address the question of how expression of GATA-1 might be differentially regulated in Sertoli and erythroid cells, we have cloned and determined the structure of the IT promoters of both the rat and mouse GATA-1 genes. The transcription regulatory mechanism(s) controlling the synthesis of exon IT-derived mRNA was investigated by transfection of wild-type and mutant reporter genes, with and without co-transfected GATA factor expression plasmids, into either fibroblasts or Sertoli cell lines. Two GATA binding sites in the IT promoter were found to be required for GATA factor-mediated activation in fibroblasts: GATA-IT-directed reporter gene expression was activated only after co-transfection with GATA-1, implying that transcriptional activation of GATA-1 in the testis might be at least partially mediated through these GATA regulatory elements. We also found that the endogenous GATA-1 gene was silent in primary culture and two different Sertoli cell lines, and that the repression of co-transfected GATA-1 reporter genes could not be relieved by forced expression of GATA-1 in Sertoli cells. Thus the GATA-IT promoter may be under the control of a regulatory network in Sertoli cells which involves both positive and negative regulation of transcription, and conserved GATA motifs found in the IT promoter may be required for transducing these effects.

Animals↗
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↗
PubMed1997

Surgical versus endoscopic correction of vesicoureteral reflux in children with neurogenic bladder dysfunction.

PURPOSE: We attempted to compare the efficacy of subureteral polytetrafluoroethylene injection and ureteral reimplantation for treating vesicoureteral reflux in children with neurogenic bladder dysfunction. MATERIALS AND METHODS: We retrospectively studied the records of all children with neurogenic bladder dysfunction and reflux into single collecting systems treated with cross-trigonal ureteroneocystostomy or subureteral polytetrafluoroethylene injection. Followup cystography was performed 2 months postoperatively and ultrasound was done twice yearly thereafter. Urodynamic data were evaluated when available. Success was defined as complete eradication of reflux. Data were stratified by procedure, patient sex, grade and laterality of reflux, and the presence of unilateral or bilateral reflux, and then compared using chi-square analysis. RESULTS: Of 85 patients (118 ureters) identified followup data were available in 95%. Subureteral polytetrafluoroethylene injection and ureteroneocystostomy were done on 60 and 47 ureters, respectively. Both groups were similar in mean patient age, followup and preoperative degree of reflux. Success rates after ureteroneocystostomy and a single injection were 84.3 versus 56.7%. The cumulative success rate of subureteral polytetrafluoroethylene injection was 61% after a second injection. The success rate of ureteroneocystostomy was significantly greater than that of injection (p = 0.02). Reflux resolved in all patients in whom injection failed and who underwent secondary reimplantation. Successful ureteroneocystostomy was unrelated to patient sex, reflux grade or laterality, or bilateral versus unilateral reflux. Subureteral polytetrafluoroethylene injection was more likely to fail in higher grades of reflux (p = 0.03) but success was otherwise unrelated to other parameters. Failure to correct reflux was unrelated to urodynamic findings. CONCLUSIONS: Primary open ureteral reimplantation is more effective than subureteral polytetrafluoroethylene injection for correcting reflux in children with neurogenic bladder dysfunction. Despite controversy related to the appropriate injectable substance, the relative technical simplicity, outpatient nature, rapid recovery and potential for successful secondary reimplantation support a role for subureteral polytetrafluoroethylene injection in managing reflux in these difficult cases.

Adolescent↗
PubMed1996

Mesodermal- vs. neuronal-specific expression of MafK is elicited by different promoters.

BACKGROUND: Small members of the Maf family of transcriptional regulatory proteins share similar basic-leucine zipper domains but have no intrinsic ability to activate transcription. One member of the family (MafK) has been shown to mediate both negative and positive regulation: in addition to forming a homodimer which represses transcription, MafK can also form a heterodimer with p45 (the large subunit of erythroid transcription factor NF-E2) to activate transcription. RESULTS: We examined the expression of mafK during murine development. mafK mRNA was first detected in 7.5 days post coitus (dpc) embryonic mesoderm and persisted in mesodermal derivatives (mesenchymal and haematopoietic cells) thereafter. However, around 13 dpc mafK was also strongly induced in neuronal cells and it is broadly expressed in neurones in postnatal mouse. The neuronal expression of mafK is directed by a distinct promoter located 6 kbp 3' to the mesoderm-specific promoter. mafK in neurones associates with a different partner molecule from p45. In transgenic mice, a regulatory domain in the immediate vicinity of the mesodermal promoter was found to direct mesenchymal, but not haematopoietic, expression of mafK. CONCLUSION: The cell type- and developmental stage-specific expression of MafK suggests that, in addition to its demonstrated role in erythroid transcriptional regulation, MafK also plays an important regulatory role in other mesodermally and neuroectodermally derived tissues during mouse embryonic development.

Animals↗
PubMed1996

Regulation and function of transcription factor GATA-1 during red blood cell differentiation.

The tissue-specific transcription factor GATA-1 is a key regulator of red blood cell differentiation. One seemingly contradictory aspect of GATA-1 function is that, while it is abundant in erythroid progenitor cells prior to the onset of overt differentiation, it does not significantly activate known GATA-1 target genes in those cells. To investigate the mechanisms underlying GATA-1 function during the transition from early to late erythropoiesis, we have examined its expression and activity in normal avian erythroid progenitor cells before and after induction of differentiation. In these primary progenitor cells, GATA-1 protein was predominantly located in the cytoplasm, while induction of differentiation caused its rapid relocalization to the nucleus, suggesting that nuclear translocation constitutes an important regulatory step in GATA-1 activation. As an alternative way of addressing the same question, we also ectopically expressed a GATA-1/estrogen receptor fusion protein (GATA-1/ER) in red blood cell progenitors, where nuclear translocation of, and transcriptional activation by, this hybrid factor are conditionally controlled by estrogen. We found that hormone-activated GATA-1/ER protein accelerated red blood cell differentiation, and concomitantly suppressed cell proliferation. These phenotypic effects were accompanied by a simultaneous suppression of c-myb and GATA-2 transcription, two genes thought to be involved in the proliferative capacity of hematopoietic progenitor cells. Thus, GATA-1 appears to promote differentiation in committed erythroid progenitor cells both by inducing differentiation-specific genes and by simultaneously suppressing genes involved in cell proliferation.

Animals↗
PubMed1996

The role of transcription factors in erythroid development.

Many DNA-binding proteins (transcription factors) that are expressed specifically in erythroid cells have been identified during the past 5 years, and more are yet to be discovered or fully described. These factors play key roles in the regulation of gene expression by combining to co-ordinate the synthesis of messenger RNA levels for essentially all known erythroid genes. Here we review some of the experiments describing the properties of these transcription factors, and provide a simple conceptual framework for how they might evoke either positive or negative responses by communication with the basal transcriptional apparatus (operating at the promoters of erythroid-specific genes) to either actively inhibit or amplify transcription.

Animals↗
PubMed1995

Synergistic regulation of human beta-globin gene switching by locus control region elements HS3 and HS4.

Proper tissue- and developmental stage-specific transcriptional control over the five genes of the human beta-globin locus is elicited in part by the locus control region (LCR), but the molecular mechanisms that dictate this determined pattern of gene expression during human development are still controversial. By use of homologous recombination in yeast to generate mutations in the LCR within a yeast artificial chromosome (YAC) bearing the entire human beta-globin gene locus, followed by injection of each of the mutated YACs into murine ova, we addressed the function of LCR hypersensitive site (HS) elements 3 and 4 in human beta-globin gene switching. The experiments revealed a number of unexpected properties that are directly attributable to LCR function. First, deletion of either HS3 or HS4 core elements from an otherwise intact YAC results in catastrophic disruption of globin gene expression at all erythroid developmental stages, despite the presence of all other HS elements in the YAC transgenes. If HS3 is used to replace HS4, gene expression is normal at all developmental stages. Conversely, insertion of the HS4 element in place of HS3 results in significant expression changes at every developmental stage, indicating that individual LCR HS elements play distinct roles in stage-specific beta-type globin gene activation. Although the HS4 duplication leads to alteration in the levels of epsilon- and gamma-globin mRNAs during embryonic erythropoiesis, total beta-type globin mRNA synthesis is balanced, thereby leading to the conclusion that all of the human beta-locus genes are competitively regulated. In summary, the human beta-globin HS elements appear to form a single, synergistic functional entity called the LCR, and HS3 and HS4 appear to be individually indispensable to the integrity of this macromolecular complex.

Animals↗
PubMed1995

Targeted disruption of the GATA3 gene causes severe abnormalities in the nervous system and in fetal liver haematopoiesis.

GATA-3 is one member of a growing family of related transcription factors which share a strongly conserved expression pattern in all vertebrate organisms. In order to elucidate GATA-3 function using a direct genetic approach, we have disrupted the murine gene by homologous recombination in embryonic stem cells. Mice heterozygous for the GATA3 mutation are fertile and appear in all respects to be normal, whereas homozygous mutant embryos die between days 11 and 12 postcoitum (p.c.) and display massive internal bleeding, marked growth retardation, severe deformities of the brain and spinal cord, and gross aberrations in fetal liver haematopoiesis.

Abnormalities, Multiple↗
PubMed1995

Structure and regulation of the chicken GATA-3 gene.

To elucidate the mechanisms underlying the tissue-restricted expression of GATA factor transcription, we have isolated and analyzed the genomic chicken GATA-3 (cGATA-3) locus. Structural analysis of the clones showed that the cGATA-3 gene consists of six exons which span more than 19 kb. Two trans-activating domains and two Zn finger domains of cGATA-3 were found to be encoded separately by exons 2/3 and 4/5, respectively, indicating that each functional domain of GATA-3 is encoded by a discrete exon. We have determined 1.7 kb of upstream promoter sequence and found a number of sequence motifs which match those of known transcription factor binding sites. Activities of presumptive regulatory regions of this gene were assessed by transfecting chimeric constructs into a chicken T cell line MSB-1. The results showed three features of cGATA-3 gene regulation. The basal promoter activity of the cGATA-3 gene is determined by sequences lying between -104 and -29 bp of the promoter region. The upstream region containing the GATA and CACCC elements in close proximity (-1280 to -1152) appeared to act as a negative transcriptional regulator, whereas the region -1151 to -850 acts as a positive regulator. Thus, the expression of cGATA-3 gene is under complex regulation and the mode of regulation of cGATA-3 gene expression is suggested to be different from that of GATA-1 genes.

Animals↗
PubMed1995

Differential regulation of transcription factor gene expression and phenotypic markers in developing sympathetic neurons.

We have examined the regulation of transcription factor gene expression and phenotypic markers in developing chick sympathetic neurons. Sympathetic progenitor cells first express the bHLH transcriptional regulator Cash-1 (a chicken achaete-scute homologue), followed by coordinate expression of Phox2, a paired homeodomain protein, and GATA-2, a zinc finger protein. SCG10, a pan-neuronal membrane protein, is first detected one stage later, followed by the catecholaminergic neurotransmitter enzyme tyrosine hydroxylase (TH). We have used these markers to ask two questions: (1) is their expression dependent upon inductive signals derived from the notochord or floor plate?; (2) does their sequential expression reflect a single linear pathway or multiple parallel pathways? Notochord ablation experiments indicate that the floor plate is essential for induction of GATA-2, Phox2 and TH, but not for that of Cash-1 and SCG10. Taken together these data suggest that the development of sympathetic neurons involves multiple transcriptional regulatory cascades: one, dependent upon notochord or floor plate-derived signals and involving Phox2 and GATA-2, is assigned to the expression of the neurotransmitter phenotype; the other, independent of such signals and involving Cash-1, is assigned to the expression of pan-neuronal properties. The parallel specification of different components of the terminal neuronal phenotype is likely to be a general feature of neuronal development.

Animals↗