Downstream Promoter Region Research Articles

GAGA Factor-dependent Transcription and Establishment of DNase Hypersensitivity Are Independent and Unrelated Events in Vivo

Using a Drosophila transgenic system we investigated the ability of GAGA factor, a putative anti-repressor, to modulate transcription-related events in the absence or presence of a bona fide activator, the Adf-1 transcription factor. In contrast to previous in vitro and in vivo data linking the binding of GAGA factor to the acquisition of DNase hypersensitivity at heat shock promoters, we observed that inserting multiple GAGA binding motifs adjacent to a minimal alcohol dehydrogenase (Adh) promoter led to strongly elevated embryonic transcription without creation of a promoter-associated DNase-hypersensitive (DH) site. Establishment of DNase hypersensitivity required the presence of both GAGA and Adf-1 binding sites and was accompanied by a further, synergistic increase in transcription. Because Adf-1 is capable neither of establishing a DH site nor of promoting efficient transcription by itself in embryos, it is likely that DH site formation depends on a GAGA factor-mediated binding of Adf-1 to chromatin, perhaps facilitated by a locally remodeled downstream promoter region. More generally we suggest that GAGA factor-binding sequences may operate in a promoter-specific context, with transcriptional activation, polymerase pausing, and/or DH site formation critically dependent on the nature of the sequences (and their binding partners) linked in cis.

Transcriptional regulation of fibroblast growth factor-2 expression in human astrocytes: implications for cell plasticity.

Induction of the fibroblast growth factor-2 (FGF-2) gene and the consequent accumulation of FGF-2 in the nucleus are operative events in mitotic activation and hypertrophy of human astrocytes. In the brain, these events are associated with cellular degeneration and may reflect release of the FGF-2 gene from cell contact inhibition. We used cultures of human astrocytes to examine whether expression of FGF-2 is also controlled by soluble growth factors. Treatment of subconfluent astrocytes with interleukin-1beta, epidermal or platelet-derived growth factors, 18-kDa FGF-2, or serum or direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate or adenylate cyclase with forskolin increased the levels of 18-, 22-, and 24-kDa FGF-2 isoforms and FGF-2 mRNA. Transfection of FGF-2 promoter-luciferase constructs identified a unique -555/-513 bp growth factor-responsive element (GFRE) that confers high basal promoter activity and activation by growth factors to a downstream promoter region. It also identified a separate region (-624/-556 bp) essential for PKC and cAMP stimulation. DNA-protein binding assays indicated that novel cis-acting elements and trans-acting factors mediate activation of the FGF-2 gene. Southwestern analysis identified 40-, 50-, 60-, and 100-kDa GFRE-binding proteins and 165-, 112-, and 90-kDa proteins that interacted with the PKC/cAMP-responsive region. The GFRE and the element essential for PKC and cAMP stimulation overlap with the region that mediates cell contact inhibition of the FGF-2 promoter. The results show a two-stage regulation of the FGF-2 gene: 1) an initial induction by reduced cell contact, and 2) further activation by growth factors or the PKC-signaling pathway. The hierarchic regulation of the FGF-2 gene promoter by cell density and growth factors or PKC reflects a two-stage activation of protein binding to the GFRE and to the PKC/cAMP-responsive region, respectively.

DNA Topoisomerase I and PC4 Can Interact with Human TFIIIC to Promote Both Accurate Termination and Transcription Reinitiation by RNA Polymerase III

A human TFIIIC-containing complex (operationally designated holo TFIIIC) has been isolated by immunoaffinity methods and further resolved into two components that are both required for promoter-directed transcription of the VA1 gene. One component, designated TFIIIC, contains 5 polypeptides previously ascribed to TFIIIC2 and 4 additional polypeptides that correspond to TFIIIC1. Included within the other component are factors, namely DNA topoisomerase I and PC4, previously shown to serve as coactivators for transcription by RNA polymerase II. Topoisomerase I and PC4 both enhance TFIIIC interactions with downstream promoter regions and promote multiple, but not single, round transcription by RNA polymerase III from preformed preinitiation complexes. Novel functions for holo TFIIIC in transcription elongation and accurate termination events that could be important for efficient reinitiation are also described.

Additional Organizational Features of the Murine Folylpolyglutamate Synthetase Gene: TWO REMOTELY SITUATED EXONS ENCODING AN ALTERNATE 5′ END AND PROXIMAL OPEN READING FRAME UNDER THE CONTROL OF A SECOND PROMOTER

Nucleotide sequence analysis of independently isolated clones from a mouse liver cDNA library identified two additional splice variants of folylpolyglutamate synthetase (FPGS) mRNA with novel sequence at the 5' end. These variants incorporate two new alternatives (exons A1a and A1b) of exon 1 in the murine FPGS gene which are also spliced to exon 2. Exon A1a encodes most of the 5'-untranslated region. Exon A1b encodes a downstream segment of the 5'-untranslated region, two ATG start codons, and a unique mitochondrial leader peptide as well as 15 additional amino acids of cytosolic FPGS not encoded by all previously identified (Roy, K., Mitsugi, K., and Sirotnak, F. M. (1996) J. Biol. Chem., 271, 23820-23827) splice variants. It was also found by direct sequencing of genomic fragments that although exon A1b is spliced to exon 2, these new alternatives (i.e. exons A1a and A1b) to exon 1 are found approximately 9.5 kilobases upstream from exons B1a, B1b, and B1c. Exons A1a and A1b are separated from each other by a 124-nucleotide intron. Sequencing of the region 5' to exon A1a revealed a nucleotide sequence that was promoter-like and different from the downstream promoter region in the content of putative cis-acting elements. Primer extension analysis identified a number of potential transcription start sites within the more 3' end of this region. FPGS RNA transcripts incorporating exons A1a and A1b were detected in both normal mouse tissues, particularly, liver and kidney, and also to a varying extent in tumors; FPGS RNA transcripts incorporating exons B1a, B1b, and B1c were detected mainly in tumors. Thus, transcription of the FPGS gene in this tissue-specific manner appears to reflect the different usage of alternates to exon 1 under the control of different promoters. An unusual splice variant identified infrequently in a mouse liver cDNA library was 2.67 kilobases in size and incorporated exons A1a and A1b and a segment of the downstream promoter region along with exons B1c and B1b and exons 2-15.

Effects of Integration Host Factor and DNA Supercoiling on Transcription from the ilvPG Promoter of Escherichia coli

Integration host factor (IHF) activates transcription from the ilvPG promoter by severely distorting the DNA helix in an upstream region of a supercoiled DNA template in a way that alters the structure of the DNA in the downstream promoter region and facilitates open complex formation. In this report, the in vivo and in vitro influence of DNA supercoiling on transcription from this promoter is examined. In the absence of IHF, promoter activity increases with increased DNA supercoiling. In the presence of IHF, the same increases in superhelical DNA densities result in larger increases in promoter activity until a maximal activation of 5-fold is obtained. However, the relative transcriptional activities of the promoter in the presence and absence of IHF at any given DNA superhelical density remains the same. Thus, IHF and increased DNA supercoiling activate transcription by different mechanisms. Also, IHF binds with equal affinities to its target site on linear and supercoiled DNA templates. Therefore, IHF binding does not activate transcription simply by increasing the local negative supercoiling of the DNA helix in the downstream promoter region or by differential binding to relaxed and supercoiled DNA templates.

Cloning and Characterization of the Mouse Glucokinase Gene Locus and Identification of Distal Liver-Specific DNase I Hypersensitive Sites

We cloned and characterized an 83-kb fragment of mouse genomic DNA containing the entire glucokinase (GK) gene. The 11 exons of the gene span a total distance of 49 kb, with exons 1β and 1L being separated by 35 kb. A total of 25,266 bp of DNA sequence information was determined: from ∼−9.2 to ∼+15 kb (24,195 bp), relative to the hepatocyte transcription start site, and from −335 to +736 bp (1071 bp), relative to the transcription start site in β cells. These sequences revealed that mouse GK is >94% identical to rat and human GK. Mouse hepatic GK mRNA is regulated by fasting and refeeding, as also occurs in the rat. Alignment of the upstream and downstream promoter regions of the mouse, rat, and human genes revealed several evolutionarily conserved regions that may contribute to transcriptional regulation. However, fusion gene studies in transgenic mice indicate that the conserved regions near the transcription start site in hepatocytes are themselves not sufficient for position-independent expression in liver. Analysis of the chromatin structure of a 48-kb region of the mouse gene using DNase I revealed eight liver-specific hypersensitive sites whose locations ranged from 0.1 to 36 kb upstream of the liver transcription start site. The availability of a single, contiguous DNA fragment containing the entire mouse GK gene should allow further studies of cell-spe- cific expression of GK to be performed.

δ-Aminolevulinate couples cycA transcription to changes in heme availability in Rhodobacter sphaeroides

In this paper, the response of the transcriptional control region of the Rhodobacter sphaeroides cytochrome c 2 gene, cycA, to intermediates in heme biosynthesis was studied. To determine if cycA transcription was regulated by heme availability, several precursors or analogs of tetrapyrroles were tested. Addition of δ-aminolevulinate (ALA), the first committed intermediate in heme biosynthesis, was shown to inhibit cycA transcription initiation at both the upstream and downstream promoter regions. In addition, an ALA auxotroph, which can grow in the presence of high levels of ALA, showed a 5 to 7-fold reduction in steady-state transcription from cycA::lacZYA operon fusions. To identify genetic elements responsible for negative regulation by ALA, trans-acting mutants with increased expression of cycA were isolated that were resistant to growth inhibition by the heme analog cohemin. These cohemin-resistant mutants (Chr) have elevated levels of several cycA transcripts and they contain cycA transcripts that had not previously been detected in wild-type cells. In addition, cycA transcription in the Chr mutants continues after the addition of ALA. Finally, we found that Chr mutants have increased ALA synthase activity, suggesting that synthesis of cytochrome c 2 and ALA synthase are controlled by a common gene product whose activity has been modified in these mutants. A model is presented to explain how changes in tetrapyrrole intermediates could provide an effective signal to control both cycA transcription and ALA synthase synthesis in R. sphaeroides.

Cloning and sequencing of the Bacillus megaterium spoIIA operon

The spoIIA operon of Bacillus megaterium has been cloned and the nucleotide sequence determined. The spoIIA sequence contains three open reading frames coding for putative proteins of 116 aa, 147 aa, and 253 aa; the first and the third genes are preceded by a ribosomal binding site. The genes are in the same order as those of B subtilis and B licheniformis. The deduced amino acid sequences of these three open reading frames show 78–92% homology with SpoIIAA, SpoIIAB and SpoIIAC of B subtilis and B licheniformis. Northern hybridization revealed that B megaterium also has two spoIIA transcripts, 1.77 kb and 2.92 kb, attaining maximum expression at t 1 and t 3, respectively. In addition, homology to a possible penicillin binding protein gene upstream and the first part of a spoVA operon downstream has been identified on the 3.34-kb fragment. The spoIIA and the downstream spoVA promoter regions are highly conserved among these three species. Sequence analysis of the spoVA promoter revealed a region upstream to the −35 that is highly conserved across Bacillus species.

Tissue-specific regulation of glucokinase gene expression.

Glucokinase contributes to the maintenance of blood glucose homeostasis by catalyzing the high Km phosphorylation of glucose in the liver and the pancreatic beta cell, the only two tissues known to express this enzyme. Molecular biological studies of the glucokinase gene and its products have advanced our understanding of how this gene is differentially regulated in the liver and beta cell. The production of an active glucokinase isoform is determined by both transcriptional and post-transcriptional events. Two different promoter regions that are widely separated in a single glucokinase gene are used to produce glucokinase mRNAs in the liver, pancreatic beta cell, and pituitary. The different transcription control regions are tissue-specific in their expression and are differentially regulated. In liver, glucokinase gene expression is regulated by insulin and cAMP, whereas in the beta cell it is regulated by glucose. The upstream glucokinase promoter region, which gives rise to the glucokinase mRNA in pituitary and pancreas, is structurally and functionally different from the downstream promoter region, which gives rise to the glucokinase mRNA in liver. The use of distinct promoter regions in a single glucokinase gene enables a different set of transcription factors to be utilized in the liver and islet, thus allowing a functionally similar gene product to be regulated in a manner consistent with the different functions of these two tissues. In addition, the splicing of the glucokinase pre-mRNA is regulated in a tissue-specific manner and can affect the activity of the gene product.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence conservation and structural organization of the glycerol-3-phosphate dehydrogenase promoter in mice and humans.

Cloned segments of the mouse glycerol-3-phosphate dehydrogenase (GPDH) gene, Gdc-1, were used to screen a human library. Human clones obtained spanned 25 kilobases of genomic DNA containing the human GPDH gene, GPD1. The 4 kb of sequence obtained from the 5'-flanking region and first exon of GPD1 was compared with the corresponding mouse sequence. Both sequences share a HindIII site located in what has proven to be the highly conserved 3' untranslated region of an upstream gene of unknown function, D15Kzl. The 3.6-kilobase segment of mouse DNA located between D15Kzl and Gdc-1 was provisionally termed the GPDH promoter. Alignment of the mouse promoter with the corresponding human sequence revealed two conserved domains. An upstream distal promoter region is approximately 900 base pairs in length. A downstream or proximal promoter region consists of approximately 300 base pairs immediately upstream of a TATA-like box and contains the fat-specific elements 1 and 2. Analysis of the chromatin structure of the Gdc-1 promoter revealed four DNase I-hypersensitive sites. They were present in DNA of liver and brown fat, in which GPDH expression is high, but were absent in DNA of spleen, in which GPDH expression is low. Methylation studies of the promoter showed it to be heavily methylated in sperm. However, the DNA from each adult somatic tissue had a unique distribution of nonmethylated sites and could easily be identified by its methylation pattern. These data suggest a structural model of the promoter that explains how Gdc-1 expression is differentially regulated in many types of cells.

Sequence Conservation and Structural Organization of the Glycerol-3-Phosphate Dehydrogenase Promoter in Mice and Humans

Cloned segments of the mouse glycerol-3-phosphate dehydrogenase (GPDH) gene, Gdc-1, were used to screen a human library. Human clones obtained spanned 25 kilobases of genomic DNA containing the human GPDH gene, GPD1. The 4 kb of sequence obtained from the 5'-flanking region and first exon of GPD1 was compared with the corresponding mouse sequence. Both sequences share a HindIII site located in what has proven to be the highly conserved 3' untranslated region of an upstream gene of unknown function, D15Kzl. The 3.6-kilobase segment of mouse DNA located between D15Kzl and Gdc-1 was provisionally termed the GPDH promoter. Alignment of the mouse promoter with the corresponding human sequence revealed two conserved domains. An upstream distal promoter region is approximately 900 base pairs in length. A downstream or proximal promoter region consists of approximately 300 base pairs immediately upstream of a TATA-like box and contains the fat-specific elements 1 and 2. Analysis of the chromatin structure of the Gdc-1 promoter revealed four DNase I-hypersensitive sites. They were present in DNA of liver and brown fat, in which GPDH expression is high, but were absent in DNA of spleen, in which GPDH expression is low. Methylation studies of the promoter showed it to be heavily methylated in sperm. However, the DNA from each adult somatic tissue had a unique distribution of nonmethylated sites and could easily be identified by its methylation pattern. These data suggest a structural model of the promoter that explains how Gdc-1 expression is differentially regulated in many types of cells.

DNA-binding and transcriptional properties of human transcription factor TFIID after mild proteolysis.

The existence of separable functions within the human class II general transcription factor TFIID was probed for differential sensitivity to mild proteolytic treatment. Independent of whether TFIID was bound to DNA or free in solution, partial digestion with either one of a variety of nonspecific endoproteases generated a protease-resistant protein product that retained specific DNA recognition, as revealed by DNase I footprinting. However, in contrast to native TFIID, which interacts with the adenovirus major late (ML) promoter over a very broad DNA region, partially proteolyzed TFIID interacted with only a small region of the ML promoter immediately surrounding the TATA sequence. This novel footprint was very similar to that observed with the TATA factor purified from yeast cells. Partially proteolyzed human TFIID could form stable complexes that were resistant to challenge by exogenous templates. It could also nucleate the assembly of transcription complexes on the ML promoter with an efficiency comparable to that of native TFIID, yielding similar levels of transcription initiation. These results suggest a model in which the human TFIID protein is composed of at least two different regions or polypeptides: a protease-resistant "core," which by itself is sufficient for promoter recognition and basal transcriptional levels, and a protease-sensitive "tail," which interacts with downstream promoter regions and may be involved in regulatory processes.

DNA-Binding and Transcriptional Properties of Human Transcription Factor TFIID after Mild Proteolysis

The existence of separable functions within the human class II general transcription factor TFIID was probed for differential sensitivity to mild proteolytic treatment. Independent of whether TFIID was bound to DNA or free in solution, partial digestion with either one of a variety of nonspecific endoproteases generated a protease-resistant protein product that retained specific DNA recognition, as revealed by DNase I footprinting. However, in contrast to native TFIID, which interacts with the adenovirus major late (ML) promoter over a very broad DNA region, partially proteolyzed TFIID interacted with only a small region of the ML promoter immediately surrounding the TATA sequence. This novel footprint was very similar to that observed with the TATA factor purified from yeast cells. Partially proteolyzed human TFIID could form stable complexes that were resistant to challenge by exogenous templates. It could also nucleate the assembly of transcription complexes on the ML promoter with an efficiency comparable to that of native TFIID, yielding similar levels of transcription initiation. These results suggest a model in which the human TFIID protein is composed of at least two different regions or polypeptides: a protease-resistant "core," which by itself is sufficient for promoter recognition and basal transcriptional levels, and a protease-sensitive "tail," which interacts with downstream promoter regions and may be involved in regulatory processes.

Negative regulation of the hepatitis B virus pre-S1 promoter by internal DNA sequences

Expression of the surface antigen gene (S gene) of hepatitis B virus is directed by two distinct promoter elements with markedly different activities. The upstream (pre-S1) promoter produces a 2.4-kb transcript at very low levels while the downstream (pre-S2) promoter produces an approximately 2.1-kb transcript in relative abundance. We have constructed a series of internal deletion mutants to analyze differential regulation of the two S gene promoters. We show here that expression directed by the pre-S1 promoter is negatively regulated by DNA sequences containing the downstream pre-S2 promoter region. Nuclear run-on analysis indicates this down-regulation to be at the level of transcription. Furthermore, promoter repression does not apear to be due to products of the S gene region. Deletion mutagenesis studies have permitted the localization of a 61-bp region that may be involved in the apparent down-regulation of the pre-S1 promoter. These results suggest the use of an unusual regulatory mechanism by a dipromoter gene in which an active internal promoter may preclude efficient use of an upstream promoter.

Deletion mapping the yeast TRP5 control region.

The yeast gene TRP5 is regulated by the general control system of amino acid biosynthesis. A TRP5-lacZ translational fusion was constructed in order to facilitate assay for TRP5 promoter function and regulation. The chromosomally integrated fusion was derepressed 4-5-fold by lysine limitation. Thus, the TRP5-lacZ fusion is regulated by general control. Deletions were constructed in vitro in the 5'-flanking region of cloned TRP5-lacZ. These deletions localized the promoter region to within 188 base pairs of the transcription start site. Two separable subregions of the promoter were localized by further deletion mapping. The boundaries of the promoter regions are approximately -188 to -114 and -91 to -29. The TATA box is contained within the downstream promoter region. Regulatory regions essential for general control overlap with these promoter elements. Each of two separable regulatory regions contains a copy of the TGACT repeat (Donahue, T. F., Daves, R. S., Lucchini, G., and Fink, G. R. (1983) Cell 32, 89-98) previously implicated in regulation of HIS4 by general control.