DNA binding by the ETS-domain transcription factor PEA3 is regulated by intramolecular and intermolecular protein.protein interactions.

The control of DNA binding by eukaryotic transcription factors represents an important regulatory mechanism. Many transcription factors are controlled by cis-acting autoinhibitory modules that are thought to act by blocking promiscuous DNA binding in the absence of appropriate regulatory cues. Here, we have investigated the determinants and regulation of the autoinhibitory mechanism employed by the ETS-domain transcription factor, PEA3. DNA binding is inhibited by a module composed of a combination of two short motifs located on either side of the ETS DNA-binding domain. A second type of protein, Ids, can act in trans to mimic the effect of these cis-acting inhibitory motifs and reduce DNA binding by PEA3. By using a one-hybrid screen, we identified the basic helix-loop-helix-leucine zipper transcription factor USF-1 as an interaction partner for PEA3. PEA3 and USF-1 form DNA complexes in a cooperative manner. Moreover, the formation of ternary PEA3.USF-1.DNA complexes requires parts of the same motifs in PEA3 that form the autoinhibitory module. Thus the binding of USF-1 to PEA3 acts as a switch that modifies the autoinhibitory motifs in PEA3 to first relieve their inhibitory action, and second, promote ternary nucleoprotein complex assembly.

DNA-binding activity of the transcription factor upstream stimulatory factor 1 (USF-1) is regulated by cyclin-dependent phosphorylation.

The ubiquitous transcription factor upstream stimulatory factor (USF) 1 is a member of the bzHLH (leucine zipper-basic-helix-loop-helix) family, which is structurally related to the Myc family of proteins. It plays a role in the regulation of many genes, including the cyclin B1 gene, which is active during the G2/M and M phases of the cell cycle and may also play a role in the regulation of cellular proliferation. We show that the affinity of recombinant USF-1 for DNA is greatly increased by treatment with active cyclin A2-p34(cdc2) or cyclin B1-p34(cdc2) complexes and that its interaction with DNA is dependent on p34(cdc2)-mediated phosphorylation. We have localized the phosphorylation site(s) to a region that lies outside the minimal DNA-binding domain but overlaps with the previously identified USF-specific region. Deletion studies of USF-1 suggest that amino acids 143-197 regulate DNA-binding activity in a phosphorylation-dependent manner.

Distinct effects of ATP on transcription complex formation and initiation in a yeast in vitro transcription system.

The stages and kinetics of transcription complex formation in a Saccharomyces cerevisiae in vitro transcription system were analysed using the anionic detergent sarkosyl. In contrast to findings from other systems, we were not able to distinguish between a fully formed pre-initiation complex and a 'rapid start' complex to which nucleotides were added. A further increase in resistance of transcription to sarkosyl was only observed 12 min after transcription initiation, by which time elongation was underway. Low concentrations of ATP, dATP or, surprisingly, the non-hydrolysable analogue ATPgammaS selectively stimulated transcription when present during assembly of transcription complexes, although the level of stimulation dropped when ATP was added progressively later. The effect of ATP did not correlate with the kinetics of template commitment, signifying that it functioned at a later stage than this, but prior to the full assembly of sarkosyl-resistant pre-initiation complexes. ATP also altered the sarkosyl resistance of initiating transcription complexes possibly by affecting a rate-limiting step leading to earlier appearance of elongated transcripts. This effect was antagonised by ATPgammaS, thus providing evidence that the stimulatory effect of ATP on pre-initiation complex formation and its effect on the lag between initiation and elongation phases are distinct.

The human placenta expresses transcription enhancer factor-1 but there is no correlation with the expression of placental lactogen.

A transcriptional enhancer which has a consensus binding sequence for transcription enhancer factor-1 (TEF-1) has been found 3' of the hPL(3) gene. We examined whether TEF-1 is expressed by the human placenta and whether such expression is co-ordinated with that of human placental lactogen (hPL). Probing Northern blots of total RNA from first trimester and term placenta, the choriocarcinoma-derived cell line JAr and primary cultured cytotrophoblast cells with a cDNA for TEF-1 revealed transcripts of 12-13 kb and 3-4 kb. The level of TEF-1 expression was the same in first trimester as compared with term placenta and in undifferentiated JAr as compared with differentiated cytotrophoblast cells. hPL expression was tenfold higher in term compared with first trimester placenta and, whilst detectable in cytotrophoblast cells, was undetectable in JAr cells. These data show that TEF-1 is expressed by the placenta but is not co-ordinated with hPL expression.

Insulin upstream factor 1 and a novel ubiquitous factor bind to the human islet amyloid polypeptide/amylin gene promoter.

The islet amyloid polypeptide (IAPP) gene is expressed primarily in the islet beta-cell and the peptide is co-secreted with insulin. To investigate mechanisms important in its regulation, we have used the electrophoretic mobility-shift assay and methylation interference to determine systematically sites of DNA-protein interactions in the human IAPP promoter. We identified beta-cell-specific DNA-protein complexes at three sites, each of which contained a consensus binding site for insulin upstream factor I (IUF-I). This complex was displaced with an antiserum to IUF-1, confirming that IUF-1 binds to the human IAPP promoter in vitro. We have also identified a DNA-protein complex within the region -220/-250 in both beta- and non-beta-cell lines. This region contains a motif with partial identity with the binding site for the ubiquitous transcription factor upstream stimulatory factor (USF), which binds to the human insulin promoter. However, purified USF was not able to bind to this putative site in the IAPP promoter and an oligonucleotide containing a functional USF-binding site was unable to displace binding from the IAPP oligonucleotide. Methylation interference revealed that the DNA-protein complex binds to a sequence that overlaps the USE-like sequence, and may therefore be a novel helix-loop-helix protein. These results suggest that, although both IAPP and insulin are beta-cell peptides, IAPP contains regulatory regions both common to and distinct from insulin.

A TATA-less promoter containing binding sites for ubiquitous transcription factors mediates cell type-specific regulation of the gene for transcription enhancer factor-1 (TEF-1).

TEF-1 is a tissue-specific human transcription factor which binds to and activates transcription from the SV40 early promoter and the HPV-16 E6/E7 promoter and may be involved in regulation of muscle-specific and placenta-specific gene expression. To investigate the mechanism of its tissue-specific expression, we have isolated up to 3 kilobase pairs of 5'-flanking DNA and characterized the promoter of the gene for TEF-1. Multiple transcription start sites centering on a motif similar to the initiator element (Inr) were identified. A minimal promoter, which contains no recognizable TATA element but contains an Inr, delimited at -137 base pairs had full transcriptional activity both in vivo in HeLa cells and in vitro in HeLa cell extracts. This promoter is also highly active in vitro in lymphoid cell extracts, but not in vivo in lymphoid cell lines, which do not express the endogenous TEF-1 gene. The minimal promoter, which is sufficient to direct tissue-specific expression of the TEF-1 gene in vivo, contains multiple sites which bind the ubiquitous transcription factors Sp1 and ATF-1. Mutation of the Inr completely abolished transcription from the major start site while transcription from the minor sites was slightly augmented. Inactivation of the proximal Sp1 site abolished transcription from the principle start site and increased transcription from a 5' minor start site. Insertion of a TATA box element did not qualitatively alter the pattern of start site usage which seemed to be dependent upon integrity of the upstream Sp1 site. These observations suggest a "cross-talk" between the Inr and a proximal element to fix transcription start sites, which is independent of spacing and the presence of a TATA element.

Positive and negative regulation of the human insulin gene by multiple trans-acting factors.

Tissue-specific expression of the human insulin gene is regulated by cis-acting DNA elements 5' to the transcription start site. Deletion of the 5' region of the human insulin gene between nucleotides -279 and -258 caused a 25-fold rise in transcriptional activity whereas further deletion to nucleotide -229 reduced transcription activity 25-fold. In vitro analysis of protein binding in the 5' regulatory region revealed: (i) the major positive regulatory region (-258 to -229) contains a protein-binding site (GC-II) with 75% sequence identity to a motif in the rat insulin I gene, shown to be a powerful transcriptional activator. GC-II motif-binding factors are not restricted to insulin-producing cell lines. (ii) An islet cell-specific factor binds between nucleotides -217 to -210 (CT-II motif). (iii) A region between nucleotides -153 and -127, containing two identical motifs, GG-I and GG-II was also revealed. GG-I-binding factors are ubiquitous, whereas binding to the GG-II motif is beta cell-specific. (iv) A ubiquitous factor binds to a motif between nucleotides -179 and -183, identical to a half-site for the cyclic nucleotide regulatory element. (v) The negative regulatory element between -279 and -258 contains overlapping binding sites for at least 3 protein factors, with differing cell-specific distributions and can independently down-regulate thymidine kinase promoter activity in a beta cell line.

A tissue-specific nuclear factor binds to multiple sites in the human insulin-gene enhancer.

Sequence-specific binding of proteins from an insulin-secreting cell line (RINm-5F) to the human insulin-gene 5' region were examined by gel-retardation and methylation-interference analysis. Specific binding of a nuclear factor to sites between nucleotides -210 to -217 and -77 to -84 was detected. The same binding activity was shown at an upstream site (-313 to -320) with low affinity. Studies using mutated binding-site probes delineated a sequence 5'-C(T/C)CTAATG-3' for high-affinity interactions. This binding activity was also present in another insulin-producing cell line (HIT.T15), but not in extracts from cell lines that did not express the insulin gene (HeLa, HL60). Cross-species comparisons show that this sequence element is highly conserved and may thus play an important role in the cell-specific regulation of insulin-gene transcription.

Synergistic enhancement of histamine release from rat peritoneal mast cells by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate is not reflected by corresponding changes in phospholipid turnover.

In an attempt to elucidate further the relationship between changes in phospholipid metabolism in, and histamine secretion from, purified rat peritoneal mast cells, the effects of the phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) on these responses in stimulated and unstimulated cells was investigated. TPA caused a dose-dependent increase in the incorporation of 32PO4(3-) into the mast cell phospholipids; phosphatidic acid (PA) and phosphatidylcholine (PC), but not phosphatidylinositol (PI). TPA synergistically enhanced histamine release from cells stimulated by anti-immunoglobulin E (IgE) and the calcium ionophore A23187, reducing its ED50 from 150 nM to 40 nM, but did not alter histamine release from cells stimulated by compound 48/80. The effect of TPA on the changes in 32PO4(3-) incorporation into phospholipids associated with the above secretagogues did not, however, correlate well with the observed effects on histamine secretion induced by the same secretagogues. These observations are discussed in relation to the known effects of phorbol esters upon both secretory processes and phospholipid metabolism in other tissues.