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Query: UNIPROT:P06889 (Mol)
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To investigate interactions between transcription factors on mammalian promoters, we constructed a set of 24 variations of the human HSP70 gene promoter in which six upstream sequence motifs are paired in every possible combination with four TATA motifs. These promoters were analyzed for in vivo expression, and selected constructs were examined by in vitro template commitment studies. Activation transcription factor (ATF) and CP1 showed dramatically different interactions with the factor(s) bound to the TATA region. CP1 functioned in vivo regardless of the TATA motif that it was paired with and was not capable of sequestering the core promoter complex in a template commitment assay. ATF activity was dramatically altered by changing the TATA motif, and ATF was able to sequester the core promoter complex. These data suggest that CP1 and ATF function by distinct mechanisms that differ with respect to interaction with the factor(s) at the TATA box. Factor Sp1 also appeared to function by a TATA-independent mechanism. These data imply that the ability of a factor to function is determined not only by the intrinsic properties of the factor but also by promoter context.
Mol Cell Biol 1990 Jan
PMID:Factor substitution in a human HSP70 gene promoter: TATA-dependent and TATA-independent interactions. 229 2

We have identified a sequence element that specifies the position of transcription initiation for the dihydrofolate reductase gene. Unlike the functionally analogous TATA box that directs RNA polymerase II to initiate transcription 30 nucleotides downstream, the positioning element of the dihydrofolate reductase promoter is located directly at the site of transcription initiation. By using DNase I footprint analysis, we have shown that a protein binds to this initiator element. Transcription initiated at the dihydrofolate reductase initiator element when 28 nucleotides were inserted between it and all other upstream sequences, or when it was placed on either side of the DNA helix, suggesting that there is no strict spatial requirement between the initiator and an upstream element. Although neither a single Sp1-binding site nor a single initiator element was sufficient for transcriptional activity, the combination of one Sp1-binding site and the dihydrofolate reductase initiator element cloned into a plasmid vector resulted in transcription starting at the initiator element. We have also shown that the simian virus 40 late major initiation site has striking sequence homology to the dihydrofolate reductase initiation site and that the same, or a similar, protein binds to both sites. Examination of the sequences at other RNA polymerase II initiation sites suggests that we have identified an element that is important in the transcription of other housekeeping genes. We have thus named the protein that binds to the initiator element HIP1 (Housekeeping Initiator Protein 1).
Mol Cell Biol 1990 Feb
PMID:Transcription initiation from the dihydrofolate reductase promoter is positioned by HIP1 binding at the initiation site. 230 58

A novel cis-acting regulatory element (designated BTE for basic transcription element) was found in the region proximal to the TATA sequence of the P-450c gene by the use of deletion mutations. This DNA element is considered to be involved in the basic transcription of the gene and does not show distinct enhancer activity in itself. Together with the XRE sequence (A. Fujisawa-Sehara, K. Sogawa, M. Yamane, and Y. Fujii-Kuriyama, Nucleic Acids Res. 15:4179-4191, 1987), however, this sequence is required for a high inducible expression of the P-450c gene in response to xenobiotic inducers. The BTE sequence contained the GC box consensus sequence and half of the NF-1-binding consensus or CAT box sequence, but their synthetic oligonucleotides, used as competitors in the gel mobility shift assays, did not compete with the BTE sequence for the binding protein, suggesting that the BTE sequence functions as a different recognition sequence from that for Sp1 or NF-1. Analogous sequences to BTE are found in the region proximal to the TATA sequence of other genes, especially other P-450 genes with different modes of regulation, suggesting that the BTE sequence plays a common regulatory role in basic transcription of genes including a group of the P-450 superfamily. The ubiquitous distribution of nuclear factor(s) binding to this element supports this suggestion.
Mol Cell Biol 1990 Apr
PMID:A novel cis-acting DNA element required for a high level of inducible expression of the rat P-450c gene. 232 4

The neural cell adhesion molecule (NCAM) is one of the most prevalent cell adhesion molecules in vertebrates. Its expression is subject to complex cell-type- and developmental-stage-dependent regulation. To study this regulation at the level of transcription, we analyzed the promoter region of the mouse NCAM gene. The NCAM promoter did not contain a typical TATA box. Transcription started at several sites that were used indiscriminately by different cell types, implying that the different NCAM isoforms are expressed from a single promoter. Sequences responsible for both promotion and inhibition of transcription resided within 840 base pairs upstream of the main transcriptional start site. The sequence from positions -645 to -37 relative to the translation initiation site directed high levels of expression in NCAM-expressing N2A cells. The same fragment was six times less active but still significantly active in L cells, but this activity was repressed by inclusion of an additional upstream segment. We mapped eight domains of interactions with nuclear proteins within the 840-base-pair region. The segment with maximum promoter activity contained two adjacent footprints, the occupation of which appeared to be mutually exclusive. One of them corresponded to an Sp1-factor-binding consensus site, the other one bound a factor with nuclear factor I activity. The single protected domain in the fragment harboring a repressor activity consisted of a GGA repeat resembling negative regulatory elements in other promoters. Three adjacent binding sites occupied an A + T-rich segment and contained ATTA motifs also found in the recognition elements of homeodomain proteins. These results show that negative and positive elements interact to regulate the tissue-specific patterns of expression of the NCAM gene and indicate that a factor related to nuclear factor I is involved in its transcriptional control.
Mol Cell Biol 1990 May
PMID:Identification of positive and negative regulatory elements governing cell-type-specific expression of the neural cell adhesion molecule gene. 232 42

During spermatogenesis, several genes are expressed in a germ cell-specific manner. Previous studies have demonstrated that rat and mouse spermatogenic cells produce a 1,700-nucleotide proenkephalin RNA, while somatic cells that express the proenkephalin gene contain a 1,450-nucleotide transcript. Using cDNA cloning, RNA protection, and primer extension analyses, we showed that transcription of the rat and mouse spermatogenic-cell RNAs is initiated downstream from the proenkephalin somatic promoter in the first somatic intron (intron As). In both species, the germ cell cap site region consists of multiple start sites distributed over a length of approximately 30 base pairs. Within rat and mouse intron As, the region upstream of the germ cell cap sites is GC rich and lacks TATA sequences. A consensus binding site for the transcription factor SP1 was identified in intron As downstream of the proenkephalin germ cell cap site region. These features are characteristic of several previously described promoters that lack TATA sequences. Homologies were also identified between the proenkephalin and rat cytochrome c spermatogenic-cell promoters, including the absence of a TATA box, a multiple start site region, and several common sequences. This promoter motif thus may be shared with other genes expressed in male germ cells.
Mol Cell Biol 1990 Jul
PMID:Transcription of the rat and mouse proenkephalin genes is initiated at distinct sites in spermatogenic and somatic cells. 235 20

The protein-DNA interactions of the upstream promoter region of the human embryonic zeta-globin gene in nuclear extracts of erythroid K562 cells and nonerythroid HeLa cells were analyzed by DNase I footprinting, gel mobility shift assay, methylation interference, and oligonucleotide competition experiments. There are mainly two clusters of nuclear factor-binding sites in the zeta promoter. The proximal cluster spans the DNA sequence from -110 to -60 and consists of binding sites for CP2, Sp1, and NF-E1. NF-E1 binding is K562 specific, whereas CP2 binding is common to both types of cells. Overlapping the NF-E1- and CP2-binding sites is a hidden Sp1-binding site or CAC box, as demonstrated by binding studies of affinity-purified Sp1. In the distal promoter region at -250 to -220, another NF-E1-binding site overlaps a CAC box or Sp1-binding site. Extract-mixing experiments demonstrated that the higher affinity of NF-E1 binding excluded the binding of Sp1 in the K562 extract. NF-E1 factors could also displace prebound Sp1 molecules. Between the two clusters of multiple-factor-binding sites are sequences recognized by other factors, including zeta-globin factors 1 and 2, that are present in both HeLa and K562 extracts. We discuss the cell type-specific, competitive binding of multiple nuclear factors in terms of functional implications in transcriptional regulation of the zeta-globin gene.
Mol Cell Biol 1990 Jan
PMID:Cell type-specific protein-DNA interactions in the human zeta-globin upstream promoter region: displacement of Sp1 by the erythroid cell-specific factor NF-E1. 240 38

The transcription start site and promoter of the rat gene coding for the transcription factor NF-1 have been identified. The NF-1 promoter was fused to the chloramphenicol acetyltransferase-coding sequence, and the resulting plasmid was transcriptionally active in the HepG2 cell line. Footprinting and gel retardation analysis indicated that the transcription factor Sp1 binds to the NF-1 promoter. Mutants in the Sp1-binding site displayed a strong reduction in transcriptional activity.
Mol Cell Biol 1990 Jan
PMID:Transcription of the promoter of the rat NF-1 gene depends on the integrity of an Sp1 recognition site. 240 42

The molecular basis for the expression of rat embryonic fibroblast tropomyosin 1 and skeletal muscle beta-tropomyosin was determined. cDNA clones encoding these tropomyosin isoforms exhibit complete identity except for two carboxy-proximal regions (amino acids 189 to 213 and 258 to 284) and different 3'-untranslated sequences. The isoform-specific regions delineate the troponin T-binding domains of skeletal muscle tropomyosin. Analysis of genomic clones indicates that there are two separate loci in the rat genome that contain sequences complementary to these mRNAs. One locus is a pseudogene. The other locus contains a single gene made up of 11 exons and spans approximately 10 kilobases. Sequences common to all mRNAs were found in exons 1 through 5 (amino acids 1 to 188) and exons 8 and 9 (amino acids 214 to 257). Exons 6 and 11 are specific for fibroblast mRNA (amino acids 189 to 213 and 258 to 284, respectively), while exons 7 and 10 are specific for skeletal muscle mRNA (amino acids 189 to 213 and 258 to 284, respectively). In addition, exons 10 and 11 each contain the entire 3'-untranslated sequences of the respective mRNAs including the polyadenylation site. Although the gene is also expressed in smooth muscle (stomach, uterus, and vas deferens), only the fibroblast-type splice products can be detected in these tissues. S1 and primer extension analyses indicate that all mRNAs expressed from this gene are transcribed from a single promoter. The promoter was found to contain G-C-rich sequences, a TATA-like sequence TTTTA, no identifiable CCAAT box, and two putative Sp1-binding sites.
Mol Cell Biol 1986 Nov
PMID:Nonmuscle and muscle tropomyosin isoforms are expressed from a single gene by alternative RNA splicing and polyadenylation. 243 92

A genomic library was prepared with DNA from a genetically enriched mouse cell line in which amplified copies of the adenosine deaminase (ADA) gene account for over 5% of the genome. Overlapping cosmid clones encompassing the entire ADA structural gene were isolated from this genomic library and used for subsequent structural and functional analyses. Nuclease protection and primer extension analyses served to identify the location of multiple transcription initiation sites at the 5' end of the structural gene. Promoter activity was found by functional analyses to reside within a 240-base-pair fragment which contains the transcription initiation sites. Sequences upstream of the transcription initiation sites are very G + C rich (77%) and include a 22 nucleotide stretch of deoxyguanylate residues and two potential Sp1 transcription factor-binding sites. Comparison of the mouse and human ADA gene promoters revealed the presence of several regions that are highly conserved with regard to both sequence content and location and may represent genetic elements which are involved in ADA gene expression.
Mol Cell Biol 1986 Dec
PMID:Molecular cloning of the murine adenosine deaminase gene from a genetically enriched source: identification and characterization of the promoter region. 243 2

The distribution of binding sites for the ultimate carcinogen anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE-l) in the 5' region of the Chinese hamster ovary aprt gene has been determined. A plasmid (pGAL) containing the entire hamster aprt gene including the 3' and 5' flanking regions was inserted into the BamHI site of the multiple cloning site of pGEM so that the T7 promoter was 5' to the aprt gene. In vitro transcription of BPDE-I-modified pGAL, using the T7 RNA polymerase, revealed two prominent transcriptional stop sites. One of these sites was located in the first exon of the aprt gene, whereas the second transcriptional stop was located approximately 150 bp upstream from the translational start site. This latter region contains two perfect GC-box consensus sequences that are potential Sp1 binding sites. Using a specific laser cutting technique to map BPDE-I DNA binding sites in the 5' flanking region of the aprt gene, we found that the DNA region containing the GC-box consensus sequences was indeed a hot spot for BPDE-I modification.
Mol Carcinog 1989
PMID:Preferential modification of GC boxes by benzo[a]pyrene-7,8-diol-9,10-epoxide. 250 85


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