Genomic footprinting of a yeast tRNA gene reveals stable complexes over the 5'-flanking region.

We have shown by genomic footprinting that the 5'-flanking region of the Saccharomyces cerevisiae tRNASUP53 gene is protected from DNase I digestion. The protected region has a 5' boundary at -40 (relative to the transcription initiation site) and extends into the coding region of the gene, with a 3' boundary at approximately +15. Although the DNase I protection over this region was much greater than at the A- and B-box internal promoters, point mutations within the A or B box that reduced transcription in vitro eliminated the upstream DNase I protection. This implies that formation of a stable complex over the 5'-flanking region is dependent on interaction of the gene with transcription factor IIIC but that stability of the complex may not require continued interaction with this factor. The DNase I protection under varied growth conditions further suggested that the upstream complex is composed of two or more components. The region over the transcription initiation site (approximately +15 to -10) was less protected in stationary-phase cultures, whereas the more upstream region (approximately -10 to -40) was protected in both exponential- and stationary-phase cultures.

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Isolation and characterization of the human diacylglycerol kinase gene

Biochemical Journal ◽

10.1042/bj2940443 ◽

1993 ◽

Vol 294 (2) ◽

pp. 443-449 ◽

Cited By ~ 9

Author(s):

K Fujikawa ◽

S Imai ◽

F Sakane ◽

H Kanoh

Keyword(s):

Transcription Initiation ◽

Diacylglycerol Kinase ◽

Initiation Site ◽

Transcription Initiation Site ◽

Jurkat Cells ◽

Upstream Region ◽

Negative Element ◽

Isolation And Characterization ◽

Flanking Region ◽

Nuclease Mapping

The 80 kDa diacylglycerol kinase (DGK) is abundantly expressed in oligodendrocytes and lymphocytes but not to a detectable extent in other cells such as neurons and hepatocytes. As an initial attempt to delineate the mechanism of the transcriptional control of the DGK gene, we have cloned from a human genomic library a 22 kb genomic fragment. The genomic clone consists of the 5′-flanking region and 17 exons coding for approx. 53% of the total exons of human DGK, including those encoding EF-hand and zinc-finger regions. The translation initiation site is located in the second exon. S1 nuclease mapping and primer extension analysis of the human DGK mRNA identified a major transcription initiation site (position +1) at 264 bp upstream from the initiator ATG. In the 5′-flanking sequence we detected a single GC box at -35 but no canonical TATA and CAAT sequences. However, the sequence starting from the cap site (AGTTCCTGCCA) is very similar to the initiator element that specifies the transcription initiation site of some housekeeping genes. In addition, the 5′-upstream region contains several putative cis-elements. Jurkat and HepG2 cells were transfected with various 5′-deletion mutants of the upstream region fused to the structural gene of chloramphenicol acetyltransferase (CAT). The CAT assay revealed that among constructs containing up to 3.4 kb of the 5′-flanking region, a fragment of 263 bp from the transcription initiation site contains a basic promoter that is active in both types of cells. Moreover, the region between -263 and -850 contains a negative element that is active in HepG2 but not in Jurkat cells. This negative element may, at least in part, be responsible for the cell type-specific expression of the DGK gene.

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Gene structure and functional analysis of the human Na+/phosphate co-transporter

Biochemical Journal ◽

10.1042/bj3240927 ◽

1997 ◽

Vol 324 (3) ◽

pp. 927-934 ◽

Cited By ~ 35

Author(s):

Yutaka TAKETANI ◽

Ken-ichi MIYAMOTO ◽

Keiko TANAKA ◽

Kanako KATAI ◽

Mika CHIKAMORI ◽

...

Keyword(s):

Vitamin D ◽

Vitamin D Receptor ◽

Transcription Initiation ◽

Thyroid Hormone Receptor ◽

Direct Repeat ◽

Initiation Site ◽

Transcription Initiation Site ◽

Receptor Expression ◽

Specific Gene ◽

Flanking Region

Three λ phage clones encompassing the Na+/phosphate co-transporter (NaPi-3) gene and its 5′ flanking region were isolated from a human genomic DNA library. The gene comprises 13 exons and 12 introns and spans approx. 14 kb. All exon–intron junctions conform to the GT/AG rule. The major transcription-initiation site was determined by primer-extension analysis and is an adenosine residue 57 bp upstream of the 3′ end of the first exon. There is a typical TATA box 28 bp upstream of the major transcription-initiation site and various cis-acting elements, including a cAMP-responsive element, AP-1, AP-2 and SP-1 sites in the 5′ flanking region. This region also contains three direct-repeat-like sequences that resemble the consensus binding sequence for members of the steroid–thyroid hormone receptor superfamily, including vitamin D. Deletion analysis suggests that the region from nt-2409 to nt-1259 in the 5′ flanking region may be involved in kidney-specific gene expression. Vitamin D responsiveness of the NaPi-3 promoter was also detected in COS-7 cells co-transfected with a human vitamin D receptor expression vector. The presence of the three vitamin D receptor-responsive elements in the NaPi-3 promoter may be important in mediating the enhanced expression of the gene by 1,25-dihydroxyvitamin D3.

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Rabbit lung surfactant protein A gene: identification of a lung-specific DNase I hypersensitive site

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1992.262.6.l662 ◽

1992 ◽

Vol 262 (6) ◽

pp. L662-L671 ◽

Cited By ~ 4

Author(s):

Q. Chen ◽

V. Boggaram ◽

C. R. Mendelson

Keyword(s):

Transcription Initiation ◽

Regulatory Element ◽

Protein A ◽

Surfactant Protein ◽

Initiation Site ◽

Dnase I ◽

Transcription Initiation Site ◽

Hypersensitive Site ◽

Rabbit Lung ◽

Dnase I Hypersensitive Site

Expression of the gene encoding pulmonary surfactant protein A, SP-A, is lung specific and developmentally and hormonally regulated. Previously, we observed that SP-A gene transcription is initiated in fetal rabbit lung after day 21 of gestation and reaches maximal levels by day 28. In the present study, a cDNA specific for rabbit SP-A was used to isolate the SP-A gene from a rabbit genomic library. A 7.6-kb fragment containing the entire structural gene and approximately 380 bp of 5'-flanking DNA was isolated and characterized. The transcription initiation site, mapped by primer extension analysis, was localized 23 bp downstream of a putative TATA element. The structural gene is composed of five exons and four introns. The first exon encodes the 5'-untranslated region of the mRNA; the translation initiation site is in exon II, and exon V contains the two polyadenylation sites that give rise to the 2.0- and 3.0-kb species of SP-A mRNA. A potential adenosine 3',5'-cyclic monophosphate (cAMP)-regulatory element (CRE) was identified at -261 bp, and sequences with homology to glucocorticoid-regulatory element (GRE) half-sites were found at -150 and -190 bp upstream of the transcription initiation site and within the first intron. A DNase I hypersensitive site was identified in genomic DNA isolated from 21- and 28-day fetal and adult rabbit lung tissues. This site was mapped within the 5'-flanking region of the SP-A gene, at approximately -80 to -180 bp upstream of the transcription initiation site. The absence of this hypersensitive site in genomic DNA of liver, kidney, and heart tissues suggests that altered chromatin structure may serve a role in lung-specific SP-A gene expression. The presence of this tissue-specific DNase I hypersensitive site in lung nuclei from 21-day gestational age fetal rabbits suggests that the SP-A gene may exist in an accessible conformation prior to the time of transcription initiation.

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Glucocorticoids stimulate human sgk1 gene expression by activation of a GRE in its 5′-flanking region

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00021.2002 ◽

2002 ◽

Vol 283 (5) ◽

pp. E971-E979 ◽

Cited By ~ 53

Author(s):

Omar A. Itani ◽

Kang Z. Liu ◽

Kristyn L. Cornish ◽

Jason R. Campbell ◽

Christie P. Thomas

Keyword(s):

Transcription Initiation ◽

Collecting Duct ◽

A549 Cells ◽

Initiation Site ◽

Specific Protein ◽

Upstream Region ◽

Mobility Shift ◽

Cell Extracts ◽

Flanking Region ◽

Gel Mobility Shift

In lung and collecting duct epithelia, glucocorticoid (GC)-stimulated Na+ transport is preceded by an increase in the protein kinase sgk1, which in turn regulates the activity of the epithelial Na+ channel (ENaC). We investigated the mechanism for GC-regulated human sgk1 expression in lung and renal epithelia. sgk1 mRNA was increased in these epithelia by GCs, and this was inhibited by actinomycin D and superinduced by cycloheximide, consistent with a transcriptional effect that did not require protein synthesis. To understand the basis for transcriptional regulation, the transcription initiation site was mapped and the 5′-flanking region cloned by PCR. A 3-kb fragment of the upstream region was coupled to luciferase and transfected into A549 cells. By deletion analysis, an imperfect GC response element (GRE) was identified that was necessary and sufficient for GC responsiveness. When tested with cell extracts, a specific protein recognized by an anti-GC receptor (GR) antibody bound the GRE in gel mobility shift assays. We conclude that GCs stimulate sgk1 expression in human epithelial cells via activation of a GRE in the 5′-flanking region of sgk1.

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Complementary Roles of Yeast Rad4p and Rad34p in Nucleotide Excision Repair of Active and Inactive rRNA Gene Chromatin

Molecular and Cellular Biology ◽

10.1128/mcb.00137-08 ◽

2008 ◽

Vol 28 (24) ◽

pp. 7504-7513 ◽

Cited By ~ 17

Author(s):

Maxime Tremblay ◽

Yumin Teng ◽

Michel Paquette ◽

Raymond Waters ◽

Antonio Conconi

Keyword(s):

Nucleotide Excision Repair ◽

Transcription Initiation ◽

Excision Repair ◽

Initiation Site ◽

Dna Lesions ◽

Transcription Initiation Site ◽

Rrna Gene ◽

Open Chromatin ◽

Coding Region ◽

Nucleotide Excision

ABSTRACT Nucleotide excision repair (NER) removes a plethora of DNA lesions. It is performed by a large multisubunit protein complex that finds and repairs damaged DNA in different chromatin contexts and nuclear domains. The nucleolus is the most transcriptionally active domain, and in yeast, transcription-coupled NER occurs in RNA polymerase I-transcribed genes (rDNA). Here we have analyzed the roles of two members of the xeroderma pigmentosum group C family of proteins, Rad4p and Rad34p, during NER in the active and inactive rDNA. We report that Rad4p is essential for repair in the intergenic spacer, the inactive rDNA coding region, and for strand-specific repair at the transcription initiation site, whereas Rad34p is not. Rad34p is necessary for transcription-coupled NER that starts about 40 nucleotides downstream of the transcription initiation site of the active rDNA, whereas Rad4p is not. Thus, although Rad4p and Rad34p share sequence homology, their roles in NER in the rDNA locus are almost entirely distinct and complementary. These results provide evidences that transcription-coupled NER and global genome NER participate in the removal of UV-induced DNA lesions from the transcribed strand of active rDNA. Furthermore, nonnucleosome rDNA is repaired faster than nucleosome rDNA, indicating that an open chromatin structure facilitates NER in vivo.

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Structure and transcriptional regulation of the Nat2 gene encoding for the drug-metabolizing enzyme arylamine N-acetyltransferase type 2 in mice

Biochemical Journal ◽

10.1042/bj20030812 ◽

2003 ◽

Vol 375 (3) ◽

pp. 593-602 ◽

Cited By ~ 24

Author(s):

Sotiria BOUKOUVALA ◽

Naomi PRICE ◽

Kathryn E. PLANT ◽

Edith SIM

Keyword(s):

Transcriptional Regulation ◽

Transcription Initiation ◽

Consensus Sequence ◽

Simian Virus 40 ◽

Initiation Site ◽

Cellular Protein ◽

Transcription Initiation Site ◽

Gene Promoters ◽

Coding Region ◽

Nat2 Gene

Arylamine N-acetyltransferases (NATs) are polymorphic enzymes, well-known for their role in the metabolism of drugs and carcinogens. Mice have three NAT isoenzymes, of which NAT2 is postulated to be involved in endogenous, as well as xenobiotic, metabolism. To understand expression of the murine Nat2 gene, we have analysed its structure and transcriptional regulation. We have accurately mapped the transcription initiation site 6.5 kb upstream of the coding region of the gene, adjacent to a recently described non-coding exon. Transcription was demonstrated to start from this region in embryonic and adult liver, spleen, submaxillary gland, kidney, brain, thymus, lung and placenta, but not in the heart. Database searches and analyses of cDNA by PCR suggested alternative splicing of the single 6.2 kb intron of Nat2, and determined the position of the polyadenylation signal at 0.44 kb downstream of the coding region of the gene. Examination of the 13 kb sequence flanking the coding and non-coding exons of Nat2 revealed a single promoter, located close to the transcription-initiation site, and indicated regions likely to harbour control elements. The Nat2 promoter consists of an atypical TATA box and a Sp1 [SV40 (simian virus 40) protein 1] box identical with that found in many housekeeping gene promoters. Activity of the Nat2 promoter was severely reduced by deletion or mutation of either of these two elements, whereas the region of the Sp1 box bound cellular protein and resisted DNase I digestion. Finally, the ability of the promoter region to bind cellular protein was reduced by competition with oligonucleotides bearing the Sp1 consensus sequence.

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