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M Horikoshi

Publications and source records attributed to M Horikoshi.

At least 127 records · Page 7Linked to original sources
PubMed1988

Transcription factor ATF interacts with the TATA factor to facilitate establishment of a preinitiation complex.

The mammalian activator protein ATF stimulates transcription from the adenovirus E4 promoter by binding to multiple upstream promoter and enhancer elements. DNAase footprint analyses have revealed that there are cooperative interactions between ATF and TFIID (the mammalian TATA factor) when both are bound simultaneously to the promoter and that these interactions in turn facilitate promoter recognition by RNA polymerase II and the general initiation factors TFIIB and TFIIE. However, the complex of TFIID and the other general factors is stable following oligonucleotide-mediated dissociation of ATF from the complete preinitiation complex. These results indicate that TFIID is a direct target for ATF, that these interactions facilitate assembly of a complete preinitiation complex, and that the role of ATF might be transient.

Adenoviridae↗
PubMed1988

Analysis of the role of the transcription factor ATF in the assembly of a functional preinitiation complex.

In the accompanying paper (Horikoshi et al., 1988) the interaction between ATF and the general transcription initiation factors was analyzed by DNAase I footprinting experiments. Here, we use transcription assays to investigate the role of ATF in the assembly of a functional preinitiation complex. Addition of an oligonucleotide containing an ATF binding site inhibits E4 transcription by sequestering ATF. However, following preincubation of the E4 promoter in the nuclear extract, transcription is refractory to inhibition by the ATF oligonucleotide. Formation of this oligonucleotide-refractory complex occurs at an early stage in the overall transcription initiation reaction and is dependent upon ATF and the general transcription factors RNA polymerase II, TFIIB, and TFIID. This latter result suggests that the assembly and maintenance of a functional preinitiation complex involves cooperative interactions among the various transcription factors. The general transcription factor TFIIE, although required for transcriptional activity, is not involved in the assembly of an ATF oligonucleotide-refractory complex. Our results support the possibility that ATF may be required only transiently for assembly of a functional preinitiation complex.

Activating Transcription Factors↗
PubMed1988

Activation of the adenovirus EIIa late promoter by a single-point mutation which enhances binding of transcription factor IID.

We have converted the TACAAA sequence present at position -29 of the adenovirus EIIa late promoter into a canonical TATA box by oligonucleotide-directed mutagenesis. When linear templates were analyzed in nuclear extracts, transcription of the mutant promoter showed a 10-fold higher level of activity than that of the wild-type promoter. This increase was correlated with an increased affinity of the mutant promoter for transcription initiation factor IID. Further analyses demonstrated that the activating functions of three EIIa late upstream promoter elements (Huang, D.-H., and Roeder, R.G. (1988) Mol. Cell. Biol. 8, 1906-1914) were maintained in the mutant promoter background. These observations indicated, first, that the upstream elements did not act merely to overcome a rate-limiting initiation step imposed by an inefficient TATA element and, second, that the strength of the interaction between transcription initiation factor IID and the TATA box was directly related to promoter activity.

Adenoviridae↗
PubMed1988

Mechanism of action of a yeast activator: direct effect of GAL4 derivatives on mammalian TFIID-promoter interactions.

We have analyzed interactions between the mammalian TATA factor (TFIID) and derivatives of the yeast activator GAL4. The interaction of the TATA factor on the adenovirus E4 promoter with GAL4 binding sites adjacent to the TATA site was qualitatively altered in response to GAL4 binding. Alterations in the TFIID interactions were observed with two GAL4 derivatives that stimulated hybrid E4 promoter activity in vitro but not with a third derivative that bound to DNA but showed no activation. These results indicate that TFIID is a direct target for a GAL4 activation domain and suggest a simple general model for the activation mechanism.

Amino Acid Sequence↗
PubMed1988

Factors involved in specific transcription by mammalian RNA polymerase II: purification, genetic specificity, and TATA box-promoter interactions of TFIID.

Selective and accurate transcription of purified genes by RNA polymerase II requires multiple factors. The factor designated TFIID was purified extensively from HeLa cell nuclear extracts by using a simple and novel complementation assay. Thus, TFIID was preferentially inactivated by mild heat treatment of a nuclear extract, and supplementation of the heat-treated extract with TFIID-containing fractions restored adenovirus major late (ML) promoter-dependent transcription. By using this assay, TFIID was purified approximately 300-fold by conventional chromatographic methods. The most purified TFIID fraction was demonstrated to be required for transcription of a number of other cellular and viral class II genes. This factor showed specific interactions with both the adenovirus ML promoter and a human heat shock 70 (hsp-70) promoter. On the ML promoter, the DNase I-protected region extended from around position -40 to position +35, although some discontinuities (and associated hypersensitive sites) were apparent near the initiation site and near position +27; the upstream and downstream boundaries of the TFIID-binding site were also confirmed by exonuclease III digestion experiments. In contrast to these results, the DNase I-protected regions on the human hsp-70 promoter were confined to a smaller area that extended from positions -35 to -19. DNase I hypersensitive sites were observed in both the adenovirus ML and hsp-70 promoters, most notably in the region at position -47. These results indicate either that there are different forms of TFIID or that a single TFIID can interact differently with distinct promoters.

Adenoviridae↗
PubMed1987

Factors involved in specific transcription in mammalian RNA polymerase II. Functional analysis of initiation factors IIA and IID and identification of a new factor operating at sequences downstream of the initiation site.

Transcription from the major late promoter of adenovirus type 2 DNA (including DNA sequences from 56 nucleotides upstream to 33 nucleotides downstream of the CAP site) was reconstituted with transcription factors purified from HeLa cells. Five components, transcription factors (TF) IIA, -B, -E, -D and RNA polymerase II, were required for accurate initiation of transcription. Kinetic analyses combined with order of addition experiments suggested that TFIIA acted first during the initiation reaction and that this interaction was followed by the action of TFIID. In agreement with these conclusions, both TFIIA and TFIID were required to render a transcription reaction partially resistant to concentrations of Sarkosyl previously shown to inhibit an early step in the formation of a preinitiation complex. Related Sarkosyl studies indicated that the inferred complex was subsequently recognized by RNA polymerase II, which resulted in an increased level of Sarkosyl-resistant transcription (in the presence of TFIIA and TFIID), and that this interaction occurred independently of TFIIB and TFIIE. However, TFIIB and TFIIE were implicated, along with the other factors and RNA polymerase II, in the subsequent formation of a highly stable preinitiation complex, which was inferred from its ability to initiate (with added nucleotides) in the presence of heparin concentrations which blocked unbound factors. The identification of a new transcription factor, which was required only when viral sequences 3' to the major late promoter were part of the transcription unit, is also reported.

Adenoviridae↗
PubMed1986

Transcription factor(s) of Ehrlich ascites tumor cells having affinity to the 'TATA' box and a further upstream region of the adenovirus 2 major late gene.

A fraction containing a transcription factor(s) of RNA polymerase II was prepared from a nuclear lysate of Ehrlich ascites tumor cells and its binding to a promoter region of the adenovirus 2 major late gene was examined. Results showed that this fraction contained a factor(s) binding to two distinct regions: a region including the 'TATA' box and another region further upstream. The upstream protected region was different from that reported to be protected by a HeLa cell factor, suggesting species specificity of transcription factor(s) in DNA binding.

Adenoviruses, Human↗
PubMed1986

Growth-inhibitory effects of epidermal growth factor and overexpression of its receptors on human squamous cell carcinomas in culture.

The role of epidermal growth factor (EGF) and its receptors in human cancers was studied using 24 human cell cultures including 15 of squamous cell carcinoma (SCC) of the skin, oral cavity, and esophagus. EGF was found to inhibit the growth and colony formation of all the SCC cells at doses that are mitogenic in many other cells, including epidermal keratinocytes and dermal fibroblasts. This inhibitory effect of EGF on SCCs was specific, because EGF did not inhibit and in some cases slightly stimulated the growth of other tumor cells, such as adenocarcinomas of the stomach, cervix, and breast and sarcomas. The amounts of EGF receptors on these SCC cells were measured by immunoprecipitation of labeled proteins with anti-EGF receptor polyclonal antibody and binding assay of membrane preparations using 125I-EGF. Of 13 SCC cell cultures tested, all except 3 of esophageal SCC showed higher levels of EGF receptor than normal epidermal keratinocytes, which contain 1.5 X 10(5) binding sites/cell. In general, SCCs of the skin and oral cavity had large amounts of EGF receptor on the order of 10(6)/cell, whereas the receptor of esophageal SCCs was on the order of 10(5)/cell. Some SCC cells had about twice as many EGF receptors as A431 cells. The values for the equilibrium dissociation constant (Kd) of these cells were on the order of nM. The sensitivity to the inhibitory effect of EGF correlated well with the elevated level of EGF receptors in 12 SCC cell lines, and higher significance was obtained when data on esophageal SCCs were excluded. The present observations suggest that EGF and EGF receptors play a role in the development of SCCs.

Carcinoma, Squamous Cell↗
PubMed1985

Conformational change of DNA binding subunit of RNA polymerase II on binding to DNA.

The spatial arrangement of the subunits of RNA polymerase II from Ehrlich ascites tumor cells was investigated by measuring the sensitivity of each subunit in the native enzyme to various proteinases. The results showed that the largest two subunits (a and b) were sensitive to all the proteinases tested, whereas two smaller subunits (e and h) were resistant to these enzymes. These results suggest that in the native enzyme subunits e and h are located in the inside of RNA polymerase II, forming a core. It was also found that the conformation of the DNA binding subunit a changes when the enzyme binds to DNA, and it becomes much more susceptible to chymotryptic digestion.

Animals↗
PubMed1985

Structural relationships of the three stimulatory factors of RNA polymerase II from Ehrlich ascites tumor cells.

The structural relationships of S-II, S-II', and S-I(b) stimulatory proteins of RNA polymerase II purified from Ehrlich ascites tumor cells were investigated. From analysis of the amino acid compositions and tryptic peptide maps of these proteins labeled with radioiodinated Bolton-Hunter reagent, it was concluded that S-I(b) is a part of S-II located at either the amino- or carboxyl-terminal and that only this region mainly contains radioiodinatable amino acid residues when labeled using 125I. On chymotryptic digestion, S-II was cleaved to 21- and 18-kDa fragments in the presence of DNA. The 21-kDa fragment was found to be sufficient for stimulation of RNA polymerase II. It was suggested that S-II' is formed by phosphorylation of S-II in the domain containing the 18-kDa fragment.

Amino Acids↗
PubMed1984

Analysis of the stimulatory factor of RNA polymerase II in the initiation and elongation complex.

The interaction between RNA polymerase II and its specific stimulatory factor S-II was studied using each component purified to near homogeneity. It was found that RNA polymerase II and S-II form a complex in the absence of DNA and nucleoside triphosphates, although their interaction was weak. This interaction became stable in the presence of a template and substrates, and a stoichiometric amount of S-II was found to remain in the transcription machinery during RNA chain elongation.

Animals↗
PubMed1984

Enhancement of the frequency of initiation by a stimulatory protein of RNA polymerase II.

By use of purified RNA polymerase II, it was demonstrated that S-II, a stimulatory protein of RNA polymerase II, enhanced the frequency of initiation of transcription from discrete sites on the promoter region of the silk fibroin gene integrated in a supercoiled plasmid DNA in the presence of manganese. In the absence of S-II, RNA polymerase II preferentially initiated RNA synthesis from site +25, 25 bases downstream from the cap site. Of these initiation sites, the initiation from site +25 was not affected by S-II, suggesting that site +25 is structurally different from other initiation sites, including the cap site, and that S-II modifies the latter sites to the same structure as that of site +25. Direct interaction between S-II and DNA in the initiation complex was shown by demonstrating a conformational change of S-II on its interaction with DNA; namely, S-II in the initiation complex was as sensitive to chymotryptic digestion as S-II interacting with DNA, whereas free S-II was completely insensitive to chymotryptic digestion.

Animals↗
PubMed1983

Identification of subunits of RNA polymerase II from Ehrlich ascites tumor cells.

A procedure was developed for large scale purification of RNA polymerase II from Ehrlich ascites tumor cells. About 2 mg of purified enzyme was obtained from 800 g of wet cells. Ten subunits were identified which behaved corresponding to the enzyme activity both on DEAE-Sephadex chromatography and on glycerol density gradient centrifugation. Analysis of tryptic peptides of each subunit showed that these subunits were independent proteins and not structurally related to each other.

Animals↗
PubMed1983

Identification of the DNA binding subunit of RNA polymerase II from Ehrlich ascites tumor cells.

The DNA binding subunits of RNA polymerase II from Ehrlich ascites tumor cells were investigated in the following three ways. (1) RNA polymerase II was dissociated in urea and the binding of the dissociated subunits to DNA was investigated. (2) The RNA polymerase II: DNA complex was dissociated progressively with various concentrations of urea, and the subunits firmly attached to DNA were investigated. (3) RNA polymerase II was dissociated into subunits in a SDS-polyacrylamide gel containing urea and blotted onto a nitrocellulose filter. The filter was then incubated with 32P-nick-translated DNA to identify the DNA binding subunits. These procedures all showed that the largest subunit a of RNA polymerase II had strong affinity to DNA. It was found that a portion of subunits b and c could be recovered in DNA fraction when analyzed by procedures (1) and (2), but no significant DNA binding activity was detected when analyzed by procedure (3), suggesting that these subunits have either a much weaker affinity toward DNA compared to a or have affinity to a itself.

Animals↗