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PubMed1994

Identification and characterization of the human transforming growth factor-alpha initiator.

Eukaryotic transcription requires promoter proximal elements. For class II promoters, two such elements are the TATA box and the initiator. The promoter for the human transforming growth factor-alpha (TGF alpha) gene has been shown to lack a TATA box, yet initiate transcription at a unique site. We have identified an approximately 13-basepair sequence between -5 and +8 as a new promoter element. We call this element the TGF alpha initiator based on the following observations: 1) it is located at the transcription initiation site; 2) the promoter activity is largely reduced by either deletion or mutation of the element; and 3) mutations result in initiation upstream of the authentic start site; the TGF alpha initiator directs site-specific initiation. An electrophoretic mobility shift assay demonstrated that a protein(s) in nuclear extracts forms complexes with the TGF alpha initiator. This interaction is sequence specific and depends on nucleotides that are critical for the promoter activity in vivo. Two polypeptides, 105 and 95 kilodaltons, were detected by Southwestern blot analysis on the basis that they specifically interact with the TGF alpha initiator. The larger polypeptide, TIBP-1, was subsequently purified by a matrix-immobilized TGF alpha initiator sequence and was shown to possess the TGF alpha initiator-specific mobility shift activity and an ability to interact with the initiator when immobilized on a membrane. In summary, we identified and characterized the TGF alpha initiator, a proximal element that is important for the accurate transcription of the TGF alpha gene, and the 105-kilodalton protein that interacts with this element.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed2004

Efficient binding of NC2.TATA-binding protein to DNA in the absence of TATA.

Negative cofactor 2 (NC2) forms a stable complex with TATA-binding protein (TBP) on promoters. This prevents the assembly of transcription factor (TF) IIA and TFIIB and leads to repression of RNA polymerase II transcription. Here we have revisited the interactions of NC2.TBP with DNA. We show that NC2.TBP complexes exhibit a significantly reduced preference for TATA box sequences compared with TBP and TBP.TFIIA complexes. In chromatin immunoprecipitations, NC2 is found on a variety of human TATA-containing and TATA-less promoters. Substantial amounts of NC2 are present in a complex with TBP in bulk chromatin. A complex of NC2.TBP displays a K(D) for DNA of approximately 2 x 10(-9) m for a 35-bp major late promoter oligonucleotide. While preferentially recognizing promoter-bound TBP, NC2 also accelerates TBP binding to promoters and stabilizes TBP.DNA complexes. Our data suggest that NC2 controls TBP binding and maintenance on DNA that is largely independent of a canonical TATA sequence.

Adenoviridae↗
PubMed2005

Mutational analysis of BTAF1-TBP interaction: BTAF1 can rescue DNA-binding defective TBP mutants.

The BTAF1 transcription factor interacts with TATA-binding protein (TBP) to form the B-TFIID complex, which is involved in RNA polymerase II transcription. Here, we present an extensive mapping study of TBP residues involved in BTAF1 interaction. This shows that residues in the concave, DNA-binding surface of TBP are important for BTAF1 binding. In addition, BTAF1 interacts with residues in helix 2 on the convex side of TBP as assayed in protein-protein and in DNA-binding assays. BTAF1 drastically changes the TATA-box binding specificity of TBP, as it is able to recruit DNA-binding defective TBP mutants to both TATA-containing and TATA-less DNA. Interestingly, other helix 2 interacting factors, such as TFIIA and NC2, can also stabilize mutant TBP binding to DNA. In contrast, TFIIB which interacts with a distinct surface of TBP does not display this activity. Since many proteins contact helix 2 of TBP, this provides a molecular basis for mutually exclusive TBP interactions and stresses the importance of this structural element for eukaryotic transcription.

Binding Sites↗
PubMed1992

Multiple DNA elements responsible for transcriptional regulation of the ornithine decarboxylase gene by protein kinase A.

Transcription of the ornithine decarboxylase (ODC) gene is rapidly elevated by activation of protein kinase A (PKA). The additive influence of three cis-acting elements is responsible for this regulation in an adrenal carcinoma cell line. Two sites, CRE2 at -48 base pairs (bp) relative to the start of transcription and CRE3 at +95 bp, are identical to the core motif of the cAMP-responsive element (CRE) of the somatostatin gene and are conserved in the mouse, rat, and human ODC genes. Mutation of CRE2 resulted in a substantial decrease in basal promoter activity, as well as a 5-fold decrease in inducibility of the ODC promoter by PKA. CRE3 did not contribute to the basal activity of the ODC promoter, but mutation of this site resulted in a 2-fold decrease in inducibility by PKA. Deletion of a 45-bp sequence (GC-box) located 5' of CRE2, also resulted in a 2-fold decrease in inducibility of the ODC promoter. DNase I protection revealed the presence of protein binding at CRE2, the TATA box, and the GC-box of the ODC promoter. Mutation of CRE2 resulted in loss of protection of this sequence, as well as the 3' extension of the footprint over the TATA box, without affecting interactions at the GC box. Antibodies to the well characterized CRE-binding protein CREB recognized proteins binding to CRE2, suggesting that binding of CREB, or an antigenically related protein, is important for the activity of CRE2. Additionally, recombinant CREB bound to a DNA probe containing the CRE2 sequence.

Adrenal Gland Neoplasms↗
PubMed2001

Transcription factor RF2a alters expression of the rice tungro bacilliform virus promoter in transgenic tobacco plants.

The promoter from rice tungro bacilliform badnavirus (RTBV) is expressed only in phloem tissues in transgenic rice plants. RF2a, a b-Zip protein from rice, is known to bind to the Box II cis element near the TATA box of the promoter. Here, we report that the full-length RTBV promoter and a truncated fragment E of the promoter, comprising nucleotides -164 to +45, result in phloem-specific expression of beta-glucuronidase (GUS) reporter genes in transgenic tobacco plants. When a fusion gene comprising the cauliflower mosaic virus 35S promoter and RF2a cDNA was coexpressed with the GUS reporter genes, GUS activity was increased by 2-20-fold. The increase in GUS activity was positively correlated with the amount of RF2a, and the expression pattern of the RTBV promoter was altered from phloem-specific to constitutive. Constitutive expression of RF2a did not induce morphological changes in the transgenic plants. In contrast, constitutive overexpression of the b-ZIP domain of RF2a had a strong effect on the development of transgenic plants. These studies suggest that expression of the b-Zip domain can interfere with the function of homologues of RF2a that regulate development of tobacco plants.

Agrobacterium tumefaciens↗
PubMed1997

Transcriptional regulation of the human erythroid 5-aminolevulinate synthase gene. Identification of promoter elements and role of regulatory proteins.

We have characterized the 5'-flanking region of the human erythroid-specific 5-amino levulinate synthase (ALAS) gene (the ALAS2 gene) and shown that the first 300 base pairs of promoter sequence gives maximal expression in erythroid cells. Transcription factor binding sites clustered within this promoter sequence include GATA motifs and CACCC boxes, critical regulatory sequences of many erythroid cell-expressed genes. GATA sites at -126/-121 (on the noncoding strand) and -102/-97 were each recognized by GATA-1 protein in vitro using erythroid cell nuclear extracts. Promoter mutagenesis and transient expression assays in erythroid cells established that both GATA-1 binding sites were functional and exogenously expressed GATA-1 increased promoter activity through these sites in transactivation experiments. A noncanonical TATA sequence at the expected TATA box location (-30/-23) bound GATA-1- or TATA-binding protein (TBP) in vitro. Conversion of this sequence to a canonical TATA box reduced expression in erythroid cells, suggesting a specific role for GATA-1 at this site. However, expression was also markedly reduced when the -30/-23 sequence was converted to a consensus GATA-1 sequence (that did not bind TBP in vitro), suggesting that a functional interaction of both factors with this sequence is important. A sequence comprising two overlapping CACCC boxes at -59/-48 (on the noncoding strand) was demonstrated by mutagenesis to be functionally important. This CACCC sequence bound Sp1, erythroid Krüppel-like factor, and basic Krüppel-like factor in vitro, while in transactivation experiments erythroid Krüppel-like factor activated ALAS2 promoter expression through this sequence. A sequence at -49/-39 with a 9/11 match to the consensus for the erythroid specific factor NF-E2 was not functional. Promoter constructs with 5'-flanking sequence from 293 base pairs to 10.3 kilobase pairs expressed efficiently in COS-1 cells as well as in erythroid cells, indicating that an enhancer sequence located elsewhere or native chromatin structure may be required for the tissue-restricted expression of the gene in vivo.

5-Aminolevulinate Synthetase↗
PubMed1990

A single DNA-binding transcription factor is sufficient for activation from a distant enhancer and/or from a promoter position.

Typical cell type-specific or inducible mammalian genes are under the control of one or more remote enhancers which transmit their effect to the promoter region located at the initiation site of transcription. Both enhancers and promoters are composed of multiple binding sites for transcription factors. To study the requirements for promoter and enhancer function, we have used a reporter gene that is completely dependent on a single DNA-binding transcription factor in vivo. This factor is a truncated, hormone-independent form of the glucocorticoid receptor which interacts strongly with a palindromic binding site. After transfection into HeLa cells, transcription of a reporter gene with one, two or four copies of the binding site upstream of the TATA box is enhanced less than 10, at least 100 and greater than 1000-fold respectively, in the presence of the receptor. Even when the TATA box is deleted, the four upstream binding sites confer receptor-dependent transcription, though from scattered initiation sites. When four copies of the palindromic binding site are placed downstream of the transcription unit, they form a very strong receptor-dependent enhancer. This enhancer can activate comparably well promoters containing binding sites for either glucocorticoid receptor, Sp1 factor, or octamer factor. Our data show that a single defined DNA-binding factor can mediate both promoter and enhancer activity, and that it can co-operate functionally both with itself and with seemingly unrelated transcription factors.

Animals↗
PubMed1994

A TATA sequence-dependent transcriptional repressor activity associated with mammalian transcription factor IIA.

In the process of characterizing cellular proteins that modulate basal transcription by RNA polymerase II, we identified a novel repressor activity specific for promoters containing consensus TATA boxes. This activity strongly represses TATA-binding protein (TBP)-dependent transcription initiation from core promoter elements containing a consensus TATA sequence, but activates TBP-dependent transcription from core promoter elements lacking a consensus TATA sequence. Purification of this activity to near homogeneity from rat liver nuclear extracts led to the surprising discovery that it co-purifies closely with mammalian transcription factor IIA (TFIIA). The close association of TATA sequence-dependent transcriptional repressor activity with TFIIA adds a new and unexpected dimension to the already complex picture of this factor's function in transcription by RNA polymerase II.

Animals↗
PubMed2002

Analysis of cis-sequence of subgenomic transcript promoter from the Figwort mosaic virus and comparison of promoter activity with the cauliflower mosaic virus promoters in monocot and dicot cells.

A sub-genomic transcript (Sgt) promoter was isolated from the Figwort mosaic virus (FMV) genomic clone. The FMV Sgt promoter was linked to heterologous coding sequences to form a chimeric gene construct. The 5'-3'-boundaries required for maximal activity and involvement of cis-sequences for optimal expression in plants were defined by 5'-, 3'-end deletion and internal deletion analysis of FMV Sgt promoter fragments coupled with a beta-glucuronidase reporter gene in both transient protoplast expression experiments and in transgenic plants. A 301 bp FMV Sgt promoter fragment (sequence -270 to +31 from the transcription start site; TSS) provided maximum promoter activity. The TSS of the FMV Sgt promoter was determined by primer extension analysis using total RNA from transgenic plants developed for FMV Sgt promoter: uidA fusion gene. An activator domain located upstream of the TATA box at -70 to -100 from TSS is absolutely required for promoter activity and its function is critically position-dependent with respect to TATA box. Two sequence motifs AGATTTTAAT (coordinates -100 to -91) and GTAAGCGC (coordinates -80 to -73) were found to be essential for promoter activity. The FMV Sgt promoter is less active in monocot cells; FMV Sgt promoter expression level was about 27.5-fold higher in tobacco cells compared to that in maize cells. Comparative expression analysis of FMV Sgt promoter with cauliflower mosaic virus (CaMV) 35S promoter showed that the FMV Sgt promoter is about 2-fold stronger than the CaMV 35S promoter. The FMV Sgt promoter is a constitutive promoter; expression level in seedlings was in the order: root>leaf>stem.

Base Sequence↗
PubMed1993

Co-crystal structure of TBP recognizing the minor groove of a TATA element.

The three-dimensional structure of a TATA-box binding polypeptide complexed with the TATA element of the adenovirus major late promoter has been determined by X-ray crystallography at 2.25 A resolution. Binding of the saddle-shaped protein induces a conformational change in the DNA, inducing sharp kinks at either end of the sequence TATAAAAG. Between the kinks, the right-handed double helix is smoothly curved and partially unwound, presenting a widened minor groove to TBP's concave, antiparallel beta-sheet. Side-chain/base interactions are restricted to the minor groove, and include hydrogen bonds, van der Waals contacts and phenylalanine-base stacking interactions.

Adenoviridae↗
PubMed2006

Transcriptional repression of human immunodeficiency virus type 1 by AP-4.

Elucidation of the mechanism of transcriptional silencing of human immunodeficiency virus type 1 (HIV-1) provirus in latently infected cells is crucial to understand the pathophysiology of HIV-1 infection and to develop novel therapies. Here we demonstrate that AP-4 is responsible for the transcriptional repression of HIV-1. We found that AP-4 site within the viral long terminal repeat (LTR) is well conserved in the majority of HIV-1 subtypes and that AP-4 represses HIV-1 gene expression by recruiting histone deacetylase (HDAC) 1 as well as by masking TATA-binding protein to TATA box. AP-4-mediated transcriptional repression was inhibited by an HDAC inhibitor, tricostatin A, and could be exerted even at distant locations from the TATA box. In addition, AP-4 interacted with HDAC1 both in vivo and in vitro. Moreover, chromatin immunoprecipitation assays have revealed that AP-4 and HDAC1 are present in the HIV-1 LTR promoter in latently infected ACH2 and U1 cells, and they are dissociated from the promoter concomitantly with the association of acetylated histone H3, TBP, and RNA polymerase II upon TNF-alpha stimulation of HIV-1 replication. Furthermore, when AP-4 is knocked down by siRNA, HIV-1 production was greatly augmented in cells transfected with a full-length HIV-1 clone. These results suggest that AP-4 may be responsible for transcriptional quiescence of latent HIV-1 provirus and give a molecular basis to the reported efficacy of combination therapy of conventional anti-HIV drugs with an HDAC inhibitor in accelerating the clearance of HIV-1 from individuals infected with the virus.

Antiviral Agents↗
PubMed1993

Isolation and characterization of the rat gene encoding glutamate dehydrogenase.

The concentration of glutamate dehydrogenase (GDH) varies strongly between different organs and between different regions within organs. To permit further studies on the regulation of GDH expression, we isolated and characterized the rat gene encoding the GDH protein. This gene contains 13 exons and spans approximately 34 kbp. The GDH gene is present as a single, autosomally located copy in the Wistar rat genome, but shows an extensive restriction-fragment-length polymorphism for several enzymes. Promoter activity of the 5'-flanking sequence is shown by transient transfection experiments. The 5'-flanking sequence contains a TTAAAA sequence at position -29, instead of a consensus TATA box and, like many other TATA-less promoters, is characterized by a very high G + C content. In addition, consensus sequences for the binding sites of the transcription factors Sp1 and Zif268 are present in the G + C-rich upstream region.

Animals↗
PubMed1992

Transcriptional regulation of the thrombomodulin gene.

The transcriptional start sites of the endogenous human thrombomodulin (TM) gene and transiently expressed TM promoter/CAT gene constructs were defined by nuclease S1 mapping which showed two closely spaced sites at +1 and +6, respectively. Transient expression and in vitro transcription assays of 5' and internal deletion mutants of the TM promoter/CAT gene constructs reveal that the region from -72 to -29 exhibits a positive acting domain which is essential for transcriptional activity, whereas the region from -373 to -225 possesses two positive acting subdomains, -343 to -277 and -245 to -225, which together augment transcriptional activity by about 40%. Electrophoretic mobility shift assays with a duplex oligonucleotide corresponding to -72 to -29 and DNase I footprinting experiments show two specific interaction products which individually or cooperatively protect the DNA sequence from about -60 to -30. These components are essential for TM gene transcription since affinity fractionation of nuclear extracts with a duplex oligonucleotide corresponding to -72 to -29 depletes the above interaction products and specifically inhibits in vitro transcription activity of the promoter, whereas addition of the eluted components specifically restores in vitro transcription activity of the promoter. Electrophoretic mobility shift assays with duplex oligonucleotides corresponding to -294 to -215, as well as -373 to -295 and DNase I footprinting experiments show two specific interaction products which individually bind to the two subdomains but not -72 to -29 and protect the coding and noncoding strands from -245 to -225, and the noncoding strand from -337 to -314, respectively. Transient expression studies reveal that the TM promoter construct starting at -51 and including the TATA box is responsive to TNF only in cell lines exhibiting sensitivity of the endogenous receptor gene to cytokine, whereas other promoter constructs possessing a TATA box sequence are insensitive to TNF in all cell types. Based upon the above data, the regulatory events involved in TNF-dependent transcriptional regulation of the TM gene can be defined with the experimental tools and conceptual framework developed by the present investigation.

Animals↗
PubMed1985

Structure of the promoter of the rat type II procollagen gene.

We have isolated several overlapping genomic clones which contain the 5' terminal portion of the rat pro-alpha 1 (II) chain gene. These clones span about 20 kilobases (kb) of contiguous DNA containing 15 kb of the gene and 5 kb of the 5' flanking sequence. Electron microscopic analysis of mRNA-DNA hybrids by R-looping shows that collectively these clones contain 16 exons which code for approximately one-third of the pro-alpha 1 (II) chain. The sizes of the exons are small, except for the first exon which is relatively large. The nucleotide sequence of the first exon and the 1000 base pairs (bp) preceding it was determined. The first exon contains a 150-bp untranslated segment and an 85-bp sequence coding for the signal peptide and a part of the NH2-terminal propeptide of type II collagen. The segment preceding the transcription initiation site contains the "TATA" box and several G + C-rich stretches, whereas the "CAT" box is not evident between -70 and -120. The hexanucleotide sequence 5'-GGGCGG-3' is found in three different places between -200 and the TATA box. The inverted complement sequence of this hexanucleotide, 5'-CCGCCC-3', is located around both -220 and -450. The hexanucleotide and its inverted sequence have been found previously in the promoter region of the tk gene of herpes virus. These sequences are known to function in a mutually dependent manner as transcription signals for the tk gene; thus, they may play a role in determining the level of transcription of this cartilage gene. The hexanucleotide, 5'-CCGCCC-3', is also found in the 21-base pair repeats of the SV40 promoter and the promoter region of hydroxymethylglutaryl-CoA reductase gene. The sequence 5'-GTGGTTAGA-3' located around -280 is identical to the "core" sequence that has been reported as enhancer element in both viral and cellular genes. These unusual structures may be related to the tissue-specific expression of this gene.

Animals↗
PubMed1985

Structure of two unlinked Drosophila melanogaster glyceraldehyde-3-phosphate dehydrogenase genes.

Two Drosophila genes that code for the enzyme glyceraldehyde-3-phosphate dehydrogenase (Gapdh) have been isolated and their structures determined by DNA sequence analysis. The two genes, Gadph-1 and Gapdh-2, are homologous to each other in their coding regions but differ entirely in the 5' and 3' flanking regions. Both genes are functionally expressed in adult flies as determined by Northern blot analysis using gene-specific probes. Gapdh-1 is mapped by in situ hybridization at position 43E-F on the right arm of the second chromosome and Gapdh-2 at position 13F on the left arm of the X chromosome. Transcription initiation sites as well as polyadenylation sites for both Gapdh transcripts have also been determined. Gapdh-1 lacks a sequence homologous to the TATA box in its -30-base pair region that is characteristic of many RNA polymerase II transcribed promoters. In contrast, Gapdh-2 contains a consensus TATA box sequence as well as a CAAT box in its promoter region. Furthermore, a sequence element ATTTGCAT (dc) and nontandem multiple direct repeats have been found in the -35 to -155-base pair 5' flanking region. Other than the intron located in the 5' noncoding region of Gapdh-2, both genes lack intervening sequences.

Amino Acid Sequence↗
PubMed1996

Genomic structure and organization of the human rBAT gene (SLC3A1).

Cystinuria is an autosomal recessive disorder of amino acid transport, manifesting as three phenotypes (I, II, and III). An amino acid transport gene, rBAT, is responsible for cystinuria. Mutation and linkage analyses have demonstrated the disease to be heterogeneous, with rBAT being the defective gene in type I cystinuria. The genomic structure of the human rBAT gene (HGMW-approved symbol SLC 3A1) has been established via two strategies: (i) construction of two different genomic libraries by subcloning the Mega-YAC921B6 (CEPH), containing rBAT, in Lambda ZAP and screening using rBAT cDNA and different PCR products; and (ii) generation and sequencing of genomic fragments by long PCR using rBAT cDNA-derived primers. The rBAT gene spans approximately 45 kb and consists of 10 exons. The introns range from 500 to 13,000 bp. All splice sites conform to the GT/AG rule. The promoter region has been further analyzed, and a predicted TATA box 98 bp upstream of the first coding ATG was identified. In addition an Alu repeat has been detected 72 bp upstream of the predicted TATA box.

Amino Acid Transport Systems, Basic↗
PubMed1986

Evidence of sequences resembling avian retrovirus long terminal repeats flanking the trout protamine gene.

Additional TATA boxes are present in the flanking regions of trout protamine genes. Their activity as promoters was assayed using an in vitro transcription system. These additional TATA boxes, together with polyadenylation signals that include the consensus AATAAA and CACTG sequences very close to the promoters, suggest that these sequences may be closely related to retroviral long terminal repeat (LTR) sequences. Other features of retroviral LTRs that are also present are short inverted repeats. The LTR-like sequences flanking the trout protamine gene show significant homology to the avian sarcoma virus LTR over a 40-bp region. The trout protamine gene falls into the relatively rare intronless class of eukaryotic genes. This suggests that the gene could have been derived from a processed gene introduced into the genome by reverse transcription of a mature mRNA. The protamine-mRNA-coding region is flanked by AACA... TGTT sequences, which might represent vestigial traces of past recombination events and whose presence supports the notion that the protamine gene sequence was of foreign origin. Recent attempts in this laboratory to transfer the protamine gene into mouse cells have resulted in a high frequency of deletions similar to those observed with constructs in which a retrovirus was used as a vector to transfect foreign DNA with promoters. The distribution of protamine genes in the animal kingdom is very sporadic, which suggests that protamine genes appeared relatively late in evolution. The nonuniform occurrence of the gene among lower vertebrates may have been the result of its horizontal transmission only to certain species, possibly by infection with retroviruses that acquired it from a different species.

Animals↗
PubMed1985

Structural and evolutionary relationships among five members of the human gamma-crystallin gene family.

We have characterized five human gamma-crystallin genes isolated from a genomic phage library. DNA sequencing of four of the genes revealed that two of them predict polypeptides of 174 residues showing 71% homology in their amino acid sequence; the other two correspond to closely related pseudogenes which contain the same in-frame termination codon at identical positions in the coding sequence. Two of the genes and one of the pseudogenes are oriented in a head-to-tail fashion clustered within 22.5 kilobases. All three contain a TATA box 60 to 80 base pairs upstream of the initiation codon and a highly conserved segment of 44 base pairs in length immediately preceding the TATA box. The two genes and the two pseudogenes are similar in structure: each contains a small 5' exon encoding three amino acids followed by two larger exons that correspond exactly to the two similar structural domains of the polypeptide. The first intron varies from 100 to 110 base pairs, and the second intron ranges from 1 to several kilobases, rendering an overall gene size of 1.7 to 4.5 kilobases. At least one of the two pseudogenes appears to have been functional before inactivation, suggesting that their identical mutation was generated by gene conversion.

Amino Acid Sequence↗