Biomedical subjects

T Leff

Publications and source records attributed to T Leff.

29 records · Page 2Linked to original sources

PubMed1990

Nuclear factors AF-1 and C/EBP bind to the human ApoB gene promoter and modulate its transcriptional activity in hepatic cells.

ApoB-100, the major protein of low density lipoprotein, is produced primarily in the liver. The apoB promoter region from -86 to -52 binds two hepatic nuclear proteins. The protein that binds to the sequence between -86 to -61, called AF-1, was characterized and also shown to interact with the apoCIII, apoA-I, and apoCII promoters. We now show that the second protein, which by DNase I footprinting binds to the sequence between -69 and -52, is the heat stable hepatic factor C/EBP. The binding of this protein is competed by a known C/EBP-binding sequence in the transthyretin gene enhancer (TRR oligo 3). In addition, the protein protects the same region in DNase I footprinting as purified C/EBP. Mutants that affect AF-1 or C/EBP binding to the apoB promoter were created. These mutants were analyzed by the gel mobility shift assay and transient transfections into HepG2 cells. We have demonstrated that the two factors bind to overlapping sites and that they modulate apoB transcriptional activity.

Apolipoprotein B-100↗

PubMed1989

A regulatory element in the ApoCIII promoter that directs hepatic specific transcription binds to proteins in expressing and nonexpressing cell types.

To better understand the mechanisms that determine cell type-specific gene expression, we have examined the transcriptional activity of a 13-nucleotide long sequence element, designated C3P, located in the promoter of the apoCIII gene. We demonstrate that this element is required for high levels of apoCIII gene expression in hepatic cells and is sufficient to determine hepatic specific expression when introduced into a heterologous promoter. A protein was identified in hepatic cell nuclear extracts, designated AF-1, that binds to this sequence and is presumably responsible for its transcriptional activity in hepatic cells. Even though the C3P element is not active in HeLa cells, a protein with C3P binding specificity was identified in HeLa cell nuclear extracts. While the HeLa protein is similar to the hepatic AF-1 in its binding specificity and relative abundance, it has approximately twice the molecular weight of the hepatic protein, indicating that they are different proteins or different forms of the same protein. A variety of murine tissue types, including those that do not express the apoCIII gene, were found to contain C3P binding proteins. We conclude that the cell type-specific activity of the C3P element is not due to the absence of C3P binding proteins in nonexpressing cells but is the result of qualitative differences in C3P binding proteins in different cell types.

Animals↗

PubMed1989

Studies of heterologous promoter trans-activation by the HTLV-II tax protein.

The tax protein of HTLV-II increases the level of steady-state mRNA produced from the HTLV-II long terminal repeat (LTR) and also activates heterologous promoters. We have previously shown that the adenovirus E3 promoter, which is trans-activated by the adenovirus E1a protein, is also trans-activated by the tax protein. To investigate the mechanism of trans-activation by tax, we analyzed E3 promoter deletion mutants to determine nucleotide sequence requirements for activation of this promoter. Our results show that removal of different upstream regions within the promoter does not result in loss of trans-activation, indicating that tax does not appear to interact with a single DNA binding protein to activate the E3 promoter. In addition, tax and E1a together activate the E3 promoter in a greater than additive fashion, suggesting that these proteins function differently. Possible mechanisms of activation by the tax protein are discussed.

Adenovirus Early Proteins↗

PubMed1988

Cell type-specific expression of the human apoB gene is controlled by two cis-acting regulatory regions.

The human apolipoprotein B (apoB) gene codes for two related proteins, apoB-100 and apoB-48. ApoB-100 is synthesized in the liver, is the major protein constituent of low density lipoprotein, and serves as the ligand for the LDL receptor. cis-acting DNA sequence elements required for hepatic specific apoB transcription were identified in hepatoma (HepG2) and epithelial carcinoma (HeLa) cell lines transfected with apoB/CAT (chloramphenicol acetyltransferase) hybrid constructions. HepG2 cells express the transfected apoB constructions at high levels relative to expression in HeLa cells. Mutational analysis of the 5'-flanking region of the apoB gene revealed the presence of positive and negative regulatory regions. The most distal of these regions, located from -261 to -128 (with respect to the start site of transcription), was found to have a roughly equivalent negative activity in both cell types. However, sequences located from -128 to -86 showed a positive activity in HepG2 cells and a negative activity in HeLa cells. Finally, a sequence element located between positions -86 and -70 was found to have a very strong positive effect in HepG2 cells and only a mild positive effect in HeLa cells. These two proximal regions located between -128 and -70 appear to act together to determine the cell type-specific expression of the apoB gene in HepG2 and HeLa cells. Using the gel mobility shift assay and the DNase I footprinting technique, we demonstrated that DNA binding proteins from HepG2 and mouse liver nuclear extracts interact with the crucial positive region located between -86 and -70. This region was also found to contain sequence elements similar to sequences found in the promoters of other apolipoprotein genes, as well as other genes that are expressed in the liver, suggesting that these genes may share some transcriptional regulatory components.

Acetyltransferases↗

PubMed1988

Expression of the human apolipoprotein E gene is regulated by multiple positive and negative elements.

Apolipoprotein E (apoE), unlike the other major lipoproteins, is synthesized in a variety of tissues. We examined which regions of the human apoE gene contributed to its tissue-specific expression using HepG2 and HeLa cells as examples of expressing and nonexpressing tissues, respectively. Regions between -360 bp and -80 bp and within the first intron were shown to be necessary for full expression activity in HepG2 cells by a nuclease protection assay which demonstrated correct transcriptional initiation of the transfected constructions. To fine map the regulatory regions, we constructed a series of deletions fused to the reporter gene chloramphenicol acetyltransferase. We discovered eight regions which had a positive effect on expression and three regions that had a negative effect on expression, in both HepG2 and HeLa cells. In addition we found three regions which had a tissue-specific negative effect on expression in HeLa cells and one region with a tissue-specific positive effect in HepG2 cells. A DNase I protection assay revealed eight footprints within the proximal 5'-flanking sequence and the first intron. Seven of these footprints fell within closely defined regions with positive expression activity. Sequence analysis of these footprint elements revealed the presence of previously identified elements and two novel elements related to each other, identified here as B1 and B2. We also defined another repeated sequence, the A element; all three of the tissue-nonspecific negative regions contained this element or sequences with homology to it. In the context of a heterologous promotor, a synthetic oligonucleotide containing the B1 and B2 elements behaved like a classical enhancer, having a positive effect on expression, even when placed at a distance. This effect was neutralized by a different synthetic oligonucleotide containing an A element repeat.

Animals↗

PubMed1988

Human apolipoprotein CIII gene expression is regulated by positive and negative cis-acting elements and tissue-specific protein factors.

Apolipoprotein CIII (apoCIII) is a major protein constituent of triglyceride-rich lipoproteins and is synthesized primarily in the liver. Cis-acting DNA elements required for liver-specific apoCIII gene transcription were identified with transient expression assays in the human hepatoma (HepG2) and epithelial carcinoma (HeLa) cell lines. In liver cells, 821 nucleotides of the human apoCIII gene 5'-flanking sequence were required for maximum levels of gene expression, while the proximal 110 nucleotides alone were sufficient. No expression was observed in similar studies with HeLa cells. The level of expression was modulated by a combination of positive and negative cis-acting sequences, which interact with distinct sets of proteins from liver and HeLa cell nuclear extracts. The proximal positive regulatory region shares homology with similarly located sequences of other genes strongly expressed in the liver, including alpha 1-antitrypsin and other apolipoprotein genes. The negative regulatory region is strikingly homologous to the human beta-interferon gene regulatory element. The distal positive region shares homology with some viral enhancers and has properties of a tissue-specific enhancer. The regulation of the apoCIII gene is complex but shares features with other genes, suggesting shuffling of regulatory elements as a common mechanism for cell type-specific gene expression.

Animals↗

PubMed1987

The human apolipoprotein C-II gene sequence contains a novel chromosome 19-specific minisatellite in its third intron.

The human apolipoprotein C-II gene was sequenced and found to contain four exons and three introns, with a major transcription initiation site located 26 base pairs downstream from a TATA sequence element. The third intron was found to be composed almost entirely of a novel 37-base pair minisatellite that is repeated six times. The minisatellite sequence was found to be present in approximately 60 different genomic locations. The minisatellite DNA sequence at three of these loci were compared and found to be highly conserved. In situ hybridization indicated that the minisatellite loci were clustered in the q13.3 band of chromosome 19. This is the first example of a chromosome- and band-specific repetitive element in the mammalian genome.

Apolipoprotein C-II↗

PubMed1986

Sequence-specific activation of transcription by adenovirus EIa products is observed in HeLa cells but not in 293 cells.

The adenovirus EIa gene products activate transcription from the viral EIII and EIIaE promoters. We studied the mechanism of this stimulation by constructing a series of chimeric promoter recombinants containing the upstream regions of the EIII and EIIaE promoters linked to the TATA box-start-site regions of the viral major late and EIIa late promoters. By introducing these recombinants into HeLa cells together with recombinants producing the EIa gene products, we demonstrated that the induction of EIII and EIIaE transcription by EIa 13S and 12S mRNA products is dependent on sequences located in the upstream region (approximately -40 to -250) of these promoters. In addition, we showed that the major late and EIIa late upstream promoter regions do not contain such EIa-responsive sequence elements. In contrast, after transfection of these chimeric promoter recombinants into 293 cells (which constitutively express the EIa proteins), we found that their relative levels of transcription are similar and markedly different from those observed when they are cotransfected into HeLa cells with EIa protein-producing recombinants. We conclude that the efficiency of transcription from a given promoter in 293 cells is not necessarily related to the presence of a specific EIa-responsive element.

Adenovirus Early Proteins↗

PubMed1985

Transcriptional analysis of the adenovirus-5 EIII promoter: absence of sequence specificity for stimulation by EIa gene products.

To identify the adenovirus-5 EIII promoter sequences that are involved in basal level of transcription, a series of promoter deletion mutants were analyzed in vivo by transfection into HeLa cells and in vitro using a HeLa whole cell extract system. Three regions within the EIII promoter were shown to be important for efficient transcription: the TATA sequence, an upstream element centered at -55/-57, and an additional element located between -111 and -233. In vivo transcriptional analysis of EIII promoter deletions in the presence of adenovirus EIa gene products have demonstrated that the same three regions are required for EIa-stimulated transcription. We conclude that there is no sequence element in the EIII promoter between -15 and -233 that is uniquely required for the stimulation of EIII transcription by EIa gene products.

Adenoviruses, Human↗

PubMed1984

Individual products of the adenovirus 12S and 13S EIa mRNAs stimulate viral EIIa and EIII expression at the transcriptional level.

Recombinant plasmids containing mutant or wild-type adenovirus serotype 2 EIa genes that produce the 12S mRNA alone, the 13S mRNA alone, or both mRNAs were cotransfected into HeLa cells with plasmids containing the viral EIIa or EIII transcription units. The amount of RNA produced from the EIIa and EIII promoters was increased by the products of both the 13S and the 12S RNAs. By measuring the level of specific transcription in nuclei isolated from transfected cells we directly demonstrate that the increased amount of EIIa RNA is due to stimulation of the rate of transcription.

Adenoviridae↗

PubMed1983

Far upstream sequences are required for efficient transcription from the adenovirus-2 E1A transcription unit.

We have investigated the requirement for sequences located upstream from the TATA box for efficient transcription from the Adenovirus-2 (Ad2) E1A promoter. A series of deletions located within the E1A promoter upstream sequences were introduced into recombinants which contain or do not contain the E1A structural sequences. The amount of E1A-specific RNA produced after transfection into HeLa cells was determined by quantitative S1 nuclease analysis. We demonstrate that sequences located more than 231 bp upstream from the E1A capsite are required for efficient transcription from the E1A promoter. However, the requirement for these stimulatory sequences is less pronounced in recombinants which contain the E1A structural sequences than in those in which these sequences have been deleted. We demonstrate also that these Ad2 stimulatory sequences activate transcription in cis when inserted upstream from the heterologous -34 to +33 Ad2 major late promoter (Ad2MLP) element which is otherwise inactive when transfected into HeLa cells. These results suggest that the 270 bp Ad2 left-terminal segment contains an enhancer-like element.

Adenoviruses, Human↗