Hepatocyte nuclear factor 6, a transcription factor that contains a novel type of homeodomain and a single cut domain.

Tissue-specific transcription is regulated in part by cell type-restricted proteins that bind to defined sequences in target genes. The DNA-binding domain of these proteins is often evolutionarily conserved. On this basis, liver-enriched transcription factors were classified into five families. We describe here the mammalian prototype of a sixth family, which we therefore call hepatocyte nuclear factor 6 (HNF-6). It activates the promoter of a gene involved in the control of glucose metabolism. HNF-6 contains two different DNA-binding domains. One of these corresponds to a novel type of homeodomain. The other is homologous to the Drosophila cut domain. A similar bipartite sequence is coded by the genome of Caenorhabditis elegans.

The yeast GAL11 protein binds to the transcription factor IIE through GAL11 regions essential for its in vivo function.

The GAL11 gene encodes an auxiliary transcription factor required for full expression of many genes in yeast. The GAL11-encoded protein (Gal11p) has recently been shown to copurify with the holoenzyme of RNA polymerase II. Here we report that Gal11p stimulates basal transcription in a reconstituted transcription system composed of recombinant or highly purified transcription factors, TFIIB, TFIIE, TFIIF, TFIIH, and TATA box-binding protein and core RNA polymerase II. We further demonstrate that each of the two domains of Gal11p essential for in vivo function respectively participates in the binding to the small and large subunits of TFIIE. The largest subunit of RNA polymerase II was coprecipitated by anti-hemagglutinin epitope antibody from crude extract of GAL11 wild type yeast expressing hemagglutinintagged small subunit of TFIIE. Such a coprecipitation of the RNA polymerase subunit was seen but in a greatly reduced amount, if extract was prepared from gal11 null yeast. In light of these findings, we suggest that Gal11p stimulates promoter activity by enhancing an association of TFIIE with the preinitiation complex in the cell.

The whn transcription factor encoded by the nude locus contains an evolutionarily conserved and functionally indispensable activation domain.

Mutations in the whn gene are associated with the phenotype of congenital athymia and hairlessness in mouse and rat. The whn gene encodes a presumptive transcription factor with a DNA binding domain of the forkhead/ winged-helix class. Two previously described null alleles encode truncated whn proteins lacking the characteristic DNA binding domain. In the rat rnu allele described here, a nonsense mutation in exon 8 of the whn gene was identified. The truncated whnrnu protein contains the DNA binding domain but lacks the 175 C-terminal amino acids of the wild-type protein. To facilitate the identification of functionally important regions in this region, a whn homolog from the pufferfish Fugu rubripes was isolated. Comparison of derived protein sequences with the mouse whn gene revealed the presence of a conserved acidic protein domain in the C terminus, in addition to the highly conserved DNA binding domain. Using fusions with a heterologous DNA binding domain, a strong transcriptional activation domain was localized to the C-terminal cluster of acidic amino acids. As the whnrnu mutant protein lacks this domain, our results indicate that a transactivation function is essential for the activity of the whn transcription factor.

TnrA, a transcription factor required for global nitrogen regulation in Bacillus subtilis.

Expression of the Bacillus subtilis nrgAB operon is derepressed during nitrogen-limited growth. We have identified a gene, tnrA, that is required for the activation of nrgAB expression under these growth conditions. Analysis of the DNA sequence of the tnrA gene revealed that it encodes a protein with sequence similarity to GlnR, the repressor of the B. subtilis glutamine synthetase operon. The tnrA mutant has a pleiotropic phenotype. Compared with wild-type cells, the tnrA mutant is impaired in its ability to utilize allantoin, gamma-aminobutyrate, isoleucine, nitrate, urea, and valine as nitrogen sources. During nitrogen-limited growth, transcription of the nrgAB, nasB, gabP, and ure genes is significantly reduced in the tnrA mutant compared with the levels seen in wild-type cells. In contrast, the level of glnRA expression is 4-fold higher in the, tnrA mutant than in wild-type cells during nitrogen restriction. The phenotype of the tnrA mutant indicates that a global nitrogen regulatory system is present in B. subtilis and that this system is distinct from the Ntr regulatory system found in enteric bacteria.

Interaction of calcineurin with a domain of the transcription factor NFAT1 that controls nuclear import.

The nuclear import of the nuclear factor of activated T cells (NFAT)-family transcription factors is initiated by the protein phosphatase calcineurin. Here we identify a regulatory region of NFAT1, N terminal to the DNA-binding domain, that controls nuclear import of NFAT1. The regulatory region of NFAT1 binds directly to calcineurin, is a substrate for calcineurin in vitro, and shows regulated subcellular localization identical to that of full-length NFAT1. The corresponding region of NFATc likewise binds calcineurin, suggesting that the efficient activation of NFAT1 and NFATc by calcineurin reflects a specific targeting of the phosphatase to these proteins. The presence in other NFAT-family transcription factors of several sequence motifs from the regulatory region of NFAT1, including its probable nuclear localization sequence, indicates that a conserved protein domain may control nuclear import of all NFAT proteins.

Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF-kappa B.

Caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives, is known to have antimitogenic, anticarcinogenic, antiinflammatory, and immunomodulatory properties. The molecular basis for these diverse properties is not known. Since the role of the nuclear factor NF-kappa B in these responses has been documented, we examined the effect of CAPE on this transcription factor. Our results show that the activation of NF-kappa B by tumor necrosis factor (TNF) is completely blocked by CAPE in a dose- and time-dependent manner. Besides TNF, CAPE also inhibited NF-kappa B activation induced by other inflammatory agents including phorbol ester, ceramide, hydrogen peroxide, and okadaic acid. Since the reducing agents reversed the inhibitory effect of CAPE, it suggests the role of critical sulfhydryl groups in NF-kappa B activation. CAPE prevented the translocation of the p65 subunit of NF-kappa B to the nucleus and had no significant effect on TNF-induced I kappa B alpha degradation, but did delay I kappa B alpha resynthesis. The effect of CAPE on inhibition of NF-kappa B binding to the DNA was specific, in as much as binding of other transcription factors including AP-1, Oct-1, and TFIID to their DNA were not affected. When various synthetic structural analogues of CAPE were examined, it was found that a bicyclic, rotationally constrained, 5,6-dihydroxy form was superactive, whereas 6,7-dihydroxy variant was least active. Thus, overall our results demonstrate that CAPE is a potent and a specific inhibitor of NF-kappa B activation and this may provide the molecular basis for its multiple immunomodulatory and antiinflammatory activities.

Overexpression of the transcription factor UBF1 is sufficient to increase ribosomal DNA transcription in neonatal cardiomyocytes: implications for cardiac hypertrophy.

The accelerated protein accumulation characteristic of cardiomyocyte hypertrophy results from increased cellular protein synthetic capacity (elevated ribosome content). The rate limiting step in ribosome accumulation is transcription of the rRNA genes. During neonatal cardiomyocyte hypertrophy induced by norepinephrine or spontaneous contraction, changes in the expression of a ribosomal DNA transcription factor, UBF, correlated with increased rates of ribosome biogenesis. We hypothesized that elevated expression of UBF was part of the mechanism by which these hypertrophic stimuli effected increases in the rate of transcription from the rDNA promoter. In this study, we have examined directly the effect of overexpressing UBF on rDNA transcription in neonatal cardiomyocytes in culture. In control experiments, a novel reporter construct for rDNA transcription (pSMECAT) showed similar increases in activity in response to hypertrophic stimuli (10(-4) M phenylephrine, 10(-7) M endothelin, and spontaneous contraction) as did the endogenous rRNA genes. When contraction-arrested cardiomyocytes were cotransfected with pSMECAT and increasing amounts of a UBF1 expression vector; a dose-dependent (3-5 fold) increase in rDNA transcription was observed. Western blot analysis confirmed that the overexpressed, FLAG-tagged UBF accumulated in the cardiomyocyte nuclei. The observation that overexpression of UBF1 is sufficient to increase rDNA transcription in neonatal cardiomyocytes provides evidence in support of the hypothesis that the regulation of UBF is a key component of the increased ribosome biogenesis and protein accumulation associated with cardiomyocyte hypertrophy.

Interaction of the 82-kDa subunit of the vaccinia virus early transcription factor heterodimer with the promoter core sequence directs downstream DNA binding of the 70-kDa subunit.

The vaccinia virus early transcription factor (VETF), a heterodimeric protein composed of 82- and 70-kDa subunits, interacts with viral early promoters at both a sequence-specific core region upstream and a sequence-independent region downstream of the RNA start site. To determine the VETF subunit-promoter interactions, 32P-labeled DNA targets were chemically synthesized with uniquely positioned phosphorothioates to which azidophenacyl bromide moieties were coupled. After incubating the derivatized promoter with VETF and exposing the complex to 302-nm light, the protein was denatured and the individual subunits with or without covalently bound DNA were isolated with specific antiserum and analyzed by SDS/polyacrylamide gel electrophoresis. Using a set of 26 duplex probes, with uniquely positioned aryl azide moieties on the coding or template strands, we found that the 82-kDa subunit interacted primarily with the core region of the promoter, whereas the 70-kDa subunit interacted with the downstream region. Nucleotide substitutions in the core region that downregulate transcription affected the binding of both subunits: the 82-kDa subunit no longer exhibited specificity for upstream regions of the promoter but also bound to downstream regions, whereas the binding of the 70-kDa subunit was abolished even though the mutations were far upstream of its binding site. These results suggested mechanisms by which the interaction of the 82-kDa subunit with the core sequence directs binding of the 70-kDa subunit to DNA downstream.

High mobility group I(Y)-like DNA-binding domains on a bacterial transcription factor.

The bacterium Myxococcus xanthus responds to blue light by producing carotenoids. It also responds to starvation conditions by developing fruiting bodies, where the cells differentiate into myxospores. Each response entails the transcriptional activation of a separate set of genes. However, a single gene, carD, is required for the activation of both light- and starvation-inducible genes. Gene carD has now been sequenced. Its predicted amino acid sequence includes four repeats of a DNA-binding domain present in mammalian high mobility group I(Y) proteins and other nuclear proteins from animals and plants. Other peptide stretches on CarD also resemble functional domains typical of eukaryotic transcription factors, including a very acidic region and a leucine zipper. High mobility group yI(Y) proteins are known to bind the minor groove of A+T-rich DNA. CarD binds in vitro an A+T-rich element that is required for the proper operation of a carD-dependent promoter in vivo.

Isolation and characterization of two human transcription factor IIH (TFIIH)-related complexes: ERCC2/CAK and TFIIH.

Transcription factor IIH (TFIIH) is a multisubunit protein complex essential for both the initiation of RNA polymerase class II (pol II)-catalyzed transcription and nucleotide excision repair of DNA. Recent studies have shown that TFIIH copurifies with the cyclin-dependent kinase (cdk)-activating kinase complex (CAK) that includes cdk7, cyclin H, and p36/MAT1. Here we report the isolation of two TFIIH-related complexes: TFIIH* and ERCC2/CAK. TFIIH* consists of a subset of the TFIIH complex proteins including ERCC3 (XPB), p62, p44, p41, and p34 but is devoid of detectable levels of ERCC2 (XPD) and CAK. ERCC2/CAK was isolated as a complex that exhibits CAK activity that cosediments with the three CAK components (cdk7, cyclin H, and p36/MAT1) as well as the ERCC2 (XPD) protein. TFIIH* can support pol II-catalyzed transcription in vitro with lower efficiency compared with TFIIH. This TFIIH*-dependent transcription reaction was stimulated by ERCC2/CAK. The ERCC2/CAK and TFIIH* complexes are each active in DNA repair as shown by their ability to complement extracts prepared from ERCC2 (XPD)- and ERCC3 (XPB)-deficient cells, respectively, in supporting the excision of DNA containing a cholesterol lesion. These data suggest that TFIIH* and ERCC2/CAK interact to form the TFIIH holoenzyme capable of efficiently assembling the pol II transcription initiation complex and directly participating in excision repair reactions.

Separation of the transcriptional coactivator and antirepression functions of transcription factor IIA.

Human transcription factor IIA (TFIIA) is composed of three subunits (alpha, beta, and gamma). TFIIA interacts with the TATA-box binding protein and can overcome repression of transcription. TFIIA was found to be necessary for VP16-mediated transcriptional activation through a coactivator function. We have separated the coactivator and antirepression activities of TFIIA. A TFIIA lacking the alpha subunit was isolated from HeLa cells. This "mini-TFIIA" interacts with the TATA-box binding protein and can overcome repression of transcription, but it is defective in transcriptional coactivator function.

The embryonic transcription factor stage specific activator protein contains a potent bipartite activation domain that interacts with several RNA polymerase II basal transcription factors.

Stage specific activator protein (SSAP) is a member of a newly discovered class of transcription factors that contain motifs more commonly found in RNA-binding proteins. Previously, we have shown that SSAP specifically binds to its recognition sequence in both the double strand and the single strand form and that this DNA-binding activity is localized to the N-terminal RNA recognition motif domain. Three copies of this recognition sequence constitute an enhancer element that is directly responsible for directing the transcriptional activation of the sea urchin late histone H1 gene at the midblastula stage of embryogenesis. Here we show that the remainder of the SSAP polypeptide constitutes an extremely potent bipartite transcription activation domain that can function in a variety of mammalian cell lines. This activity is as much as 3 to 5 times stronger than VP16 at activating transcription and requires a large stretch of amino acids that contain glutamine-glycine rich and serine-threonine-basic amino acid rich regions. We present evidence that SSAP's activation domain shares targets that are also necessary for activation by E1a and VP16. Finally, SSAP's activation domain is found to participate in specific interactions in vitro with the basal transcription factors TATA-binding protein, TFIIB, TFIIF74, and dTAF(II) 110.

The adipocyte specific transcription factor C/EBPalpha modulates human ob gene expression.

The ob gene product, leptin, apparently exclusively expressed in adipose tissue, is a signaling factor regulating body weight homeostasis and energy balance. ob gene expression is increased in obese rodents and regulated by feeding, insulin, and glucocorticoids, which supports the concept that ob gene expression is under hormonal control, which is expected for a key factor controlling body weight homeostasis and energy balance. In humans, ob mRNA expression is increased in gross obesity; however, the effects of the above factors on human ob expression are unknown. We describe the structure of the human ob gene and initial functional analysis of its promoter. The human ob gene's three exons cover approximately 15 kb of genomic DNA. The entire coding region is contained in exons 2 and 3, which are separated by a 2-kb intron. The first small 30-bp untranslated exon is located >10.5 kb upstream of the initiator ATG codon. Three kilobases of DNA upstream of the transcription start site has been cloned and characterized. Only 217 bp of 5' sequence are required for basal adipose tissue-specific expression of the ob gene as well as enhanced expression by C/EBPalpha. Mutation of the single C/EBPalpha site in this region abolished inducibility of the promoter by C/EBPalpha in cotransfection assays. The gene structure will facilitate our analysis of ob mutations in human obesity, whereas knowledge of sequence elements and factors regulating ob gene expression should be of major importance in the prevention and treatment of obesity.

Constitutively active human Notch1 binds to the transcription factor CBF1 and stimulates transcription through a promoter containing a CBF1-responsive element.

Notch is a transmembrane receptor that plays a critical role in cell fate determination. In Drosophila, Notch binds to and signals through Suppressor of Hairless. A mammalian homologue of Suppressor of Hairless, named CBF1 (or RBPJk), is a ubiquitous transcription factor whose function in mammalian Notch signaling is unknown. To determine whether mammalian Notch can stimulate transcription through a CBF1-responsive element (RE), we cotransfected a CBF1-RE-containing chloramphenicol acetyltransferase reporter and N1(deltaEC), a constitutively active form of human Notch1 lacking the extracellular domain, into DG75, COS-1, HeLa, and 293T cells, which all contain endogenous CBF1. N1(deltaEC) dramatically increased chloramphenicol acetyltransferase activity in these cells, indicating functional coupling of Notch1 and CBF1. The activity was comparable to that produced by the Epstein-Barr virus protein EBNA2, a well-characterized, potent transactivator of CBF1. To test whether CBF1 and Notch1 interact physically, we tagged CBF1 with an epitope from the influenza virus hemagglutinin or with the N-terminal domain of gal4, and transfected the tagged CBF1 plus N1(deltaEC) into COS-1 cells. Cell lysates were immunoprecipitated and immunoblotted with several anti-Notch1 antibodies [to detect N1(deltaEC)] or with antibodies to hemagglutinin or gal4 (to detect CBF1). Each immunoprecipitate contained a complex of N1(deltaEC) and CBF1. In summary, we find that the truncated, active form of human Notch1, N1(deltaEC), binds CBF1 and activates transcription through a CBF1-RE-containing promoter. We conclude that CBF1 is a critical downstream protein in the human Notch1 signaling pathway.