Gene TATA Research Articles

Effect of dihydrotestosterone on the expression of mucin 1 and the activity of Wnt signaling in mouse corneal epithelial cells.

To explore the effects of the androgen dihydrotestosterone on the expression of mucin 1 (MUC1) and the activity of Wnt signaling in mouse corneal epithelial cells. Primary mouse corneal epithelial cells were isolated from the corneas of BALB/c mice. Quantitative real-time polymerase chain reaction, immunofluorescence and Western blot analysis were used to quantify the differential expression of selected genes. The androgen receptor was silenced by transfecting cells with androgen receptor shRNAs. TOP-Flash and FOP-flash reporter plasmids were used to measure β-catenin-driven transcription. Dihydrotestosterone treatment increased MUC1 expression and activated the Wnt signaling pathway and led to the translocation of β-catenin and upregulation of the Wnt downstream target gene TATA box binding protein and urokinase plasminogen activator. These effects were prevented by downregulating the androgen receptor. Androgens may protect against dry eye by regulating the expression of MUC1 which is stimulated by the activation of Wnt signaling via the androgen receptor. An understanding of the mechanisms associated with androgen-mediated protection against dry eye is an important step in developing new therapies for this disease.

Abstract P5-06-06: Expression of APOBEC3B in primary breast cancer of Japanese women

Abstract Background: Human cancer genomes contain tens of thousands of mutations. APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) family of cytidine deaminases normally function in innate immune responses that protects against retrovirus and retrotransposon propagation. However, these enzymes can also deaminate cytosines in the host genome and generate C to T mutations. APOBEC3B is overexpressed in several human cancer types, and this overexpression correlates with the pres[not]ence of the APOBEC3B mutation signature. Recent studies have demonstrated APOBEC3B as a source of mutations in various malignancies including breast cancers. However, the relationships between the expression of APOBEC3B in breast cancer and the clinicopathological features have not been fully elucidated. Aims: To investigate the expression of APOBEC3B mRNA in primary breast cancers and to evaluate the relationships between the APOBEC3B mRNA expression and the clinicopathological characteristics and prognosis in the primary breast cancer of Japanese women. Methods: Specimens were obtained from 305 patients with primary breast cancers who underwent surgery without neoadjuvant systemic therapy. APOBEC3B mRNA expression was analysed using quantitative reverse transcription-PCR (qRT-PCR). The APOBEC3B expression level was normalized to that of the constitutive housekeeping gene TATA binding protein (TBP). Four breast cancer subtypes were determined by the immunohistochemical analysis of ER, PR and HER2; hormone receptor (HR; ER and/or PR)+/HER2-, HR+/HER2+, HR-/HER2+(HER2) and triple negative (TN). Results: Expression of APOBEC3B mRNA was detected in 277 tumors, while it was not detected in 28 tumors. The APOBEC3B expression was significantly higher in ER-negative, PR-negative, high grade tumors. The APOBEC3B expression was positively correlated with Ki67 index and highest in TN and lowest in HR+/HER2- subtype. There were no correlations between the APOBEC3B expression and age, tumor size, lymph node metastasis and the stage. The APOBEC3B expression was not statistically different between invasive ductal carcinoma and DCIS, suggesting that the APOBEC3B expression is related to the carcinogenesis of breast cancer. High expression of APOBEC3B was significantly associated with the poor recurrence free survival in all cases and ER-positive cases; however, APOBEC3B expression was not related to the prognosis in ER-negative tumors. Conclusions: The high APOBEC3B expression was related to the aggressive phenotype of breast cancer and the poor prognosis. Citation Format: Eriko Tokunaga, Nami Yamashita, Kimihiro Tanaka, Yuka Inoue, Hiroshi Saeki, Eiji Oki, Hiroyuki Kitao, Yoshihiko Maehara. Expression of APOBEC3B in primary breast cancer of Japanese women [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-06-06.

Identification of long intergenic region sequences involved in maize streak virus replication

The main cis-acting control regions for replication of the single-stranded DNA genome of maize streak virus (MSV) are believed to reside within an approximately 310 nt long intergenic region (LIR). However, neither the minimum LIR sequence required nor the sequence determinants of replication specificity have been determined experimentally. There are iterated sequences, or iterons, both within the conserved inverted-repeat sequences with the potential to form a stem-loop structure at the origin of virion-strand replication, and upstream of the rep gene TATA box (the rep-proximal iteron or RPI). Based on experimental analyses of similar iterons in viruses from other geminivirus genera and their proximity to known Rep-binding sites in the distantly related mastrevirus wheat dwarf virus, it has been hypothesized that the iterons may be Rep-binding and/or -recognition sequences. Here, a series of LIR deletion mutants was used to define the upper bounds of the LIR sequence required for replication. After identifying MSV strains and distinct mastreviruses with incompatible replication-specificity determinants (RSDs), LIR chimaeras were used to map the primary MSV RSD to a 67 nt sequence containing the RPI. Although the results generally support the prevailing hypothesis that MSV iterons are functional analogues of those found in other geminivirus genera, it is demonstrated that neither the inverted-repeat nor RPI sequences are absolute determinants of replication specificity. Moreover, widely divergent mastreviruses can trans-replicate one another. These results also suggest that sequences in the 67 nt region surrounding the RPI interact in a sequence-specific manner with those of the inverted repeat.

β-Globin Intergenic Transcription and Histone Acetylation Dependent on an Enhancer

Histone acetyltransferases are associated with the elongating RNA polymerase II (Pol II) complex, supporting the idea that histone acetylation and transcription are intertwined mechanistically in gene coding sequences. Here, we studied the establishment and function of histone acetylation and transcription in noncoding sequences by using a model locus linking the beta-globin HS2 enhancer and the embryonic epsilon-globin gene in chromatin. An intact HS2 enhancer that recruits RNA Pol II is required for intergenic transcription and histone H3 acetylation and K4 methylation between the enhancer and target gene. RNA Pol II recruitment to the target gene TATA box is not required for the intergenic transcription or intergenic histone modifications, strongly implying that they are properties conferred by the enhancer. However, Pol II recruitment at HS2, intergenic transcription, and intergenic histone modification are not sufficient for transcription or modification of the target gene: these changes require initiation at the TATA box of the gene. The results suggest that intergenic and genic transcription complexes are independent and possibly differ from one another.

RHAMM/CD168 Is a Novel Leukemia Associated Antigen with Prognostic Value for Patients with B-Cell Chronic Lymphocytic Leukemia.

Abstract Background and Aims: Differential expression of molecules in patients with B-cell chronic lymphocytic leukemia (B-CLL) might define suitable targets for T cell based vaccines and/or antibody approaches. Methods: We assessed the mRNA expression of the tumor associated antigen (TAA) RHAMM/CD168 defined earlier by serological analysis of cDNA expression libraries (SEREX) from leukemic cells. Results: Peripheral blood mononuclear cells from 40 B-CLL patients and 20 healthy volunteers (HVs) were examined by quantitative RT-PCR. A leukemia-restricted expression of the antigen RHAMM/CD168 was observed in 39/40 B-CLL patients in contrast to the absence of its expression in HVs. To evaluate the immunogenicity of this novel LAA, mixed lymphocyte peptide cultures (MLPCs), followed by enzyme-linked immunosorbent spot (ELISPOT) and flow cytometry assays were performed to detect antigen-specific CD8+ T cells. RHAMM/CD168 specific responses by CD8+ HLA-A2/R3tetramer+CCR7-CD45RAhigh effector T cells were detected. As these CD8+ T cells contribute to the elimination of RHAMM+ CLL cells, we questioned whether expression of the antigen would be associated with a better survival. RHAMM/CD168 expression revealed to be higher in patients with unmutated IgVH status. RHAMM normalized against the housekeeping gene TATA binding protein (TBP), i.e. the RHAMM/TBP ratio was defined as a prognostic surrogate marker for B-CLL. B-CLL patients with a RHAMM/TBP ratio > 1.67 showed a significantly shorter treatment free survival (TFS) (Fig. 1). A tendency towards higher RHAMM/TBP expression ratios was observed in B-CLL cases with del11q. Conclusion: RHAMM/CD168 is a novel LAA in B-CLL patients, an antigen correlating with the clinical course of the disease. Therefore, we consider RHAMM/CD168 an interesting target for immunotherapy in early stage B-CLL patients, especially with worse prognosis (IgVH unmutated). Figure 1. Treatment-free-survival (TFS) according to RHAMM/TBP ratio Figure 1. Treatment-free-survival (TFS) according to RHAMM/TBP ratio

The murine G+C-rich promoter binding protein mGPBP is required for promoter-specific transcription.

The archetypal TATA-box deficient G+C-rich promoter of the murine adenosine deaminase gene (Ada) requires a 48-bp minimal self-sufficient promoter element (MSPE) for function. This MSPE was used to isolate a novel full-length cDNA clone that encodes a 66-kDa murine G+C-rich promoter binding protein (mGPBP). The mGPBP mRNAs are ubiquitously expressed as either 3.0- or 3.5-kb forms differing in 3' polyadenylation site usage. Purified recombinant mGPBP, in the absence of any other mammalian cofactors, binds specifically to both the murine Ada gene promoter's MSPE and the nonhomologous human Topo IIalpha gene's G+C-rich promoter. In situ binding assays, immunoprecipitation, and Western blot analyses demonstrated that mGPBP is a nuclear factor that can form complexes with TATA-binding protein, TFIIB, TFIIF, RNA polymerase II, and P300/CBP both in vitro and in intact cells. In cotransfection assays, increased mGPBP expression transactivated the murine Ada gene's promoter. Sequestering of GPBP present in HeLa cell nuclear extract by immunoabsorption completely and reversibly suppressed extract-dependent in vitro transcription from the murine Ada gene's G+C-rich promoter. However, transcription from the human Topo IIalpha gene's TATA box-containing G+C-rich promoter was only partially suppressed and the adenovirus major late gene's classical TATA box-dependent promoter is totally unaffected under identical assay conditions. These results implicate GPBP as a requisite G+C-rich promoter-specific transcription factor and provide a mechanistic basis for distinguishing transcription initiated at a TATA box-deficient G+C-rich promoter from that initiated at a TATA box-dependent promoter.

A single-stranded promoter for RNA polymerase III.

Single strands of DNA serve, in rare instances, as promoters for transcription; duplex DNA promoters with individual strands that also have a promoter capacityfunction have not been described. We show that the nontranscribed strand of the Saccharomyces cerevisiae U6 snRNA gene directs transcription initiation factor IIIB-requiring and accurately initiating transcription by RNA polymerase III. The nontranscribed strand promoter is much more extended than its duplex DNA counterpart, comprising the U6 gene TATA box, a downstream T(7) tract, and an upstream-lying segment. A requirement for placement of the 3' end of the transcribed (template) strand within the confines of the transcription bubble is seen as indicating that the nontranscribed strand provides a scaffold for RNA polymerase recruitment but is deficient at a subsequent step of transcription initiation factor IIIB's direct involvement in promoter opening.

Effect of Bilirubin UDP Glucuronosyltransferase 1 Gene TATA Box Genotypes on Serum Bilirubin Concentrations in Chronic Liver Injuries

TATA box abnormality in the promoter region of the bilirubin UDP glucuronosyltransferase 1 gene has been reported to cause Gilbert's syndrome in white subjects. It has also been reported that the majority of Japanese patients with Gilbert's syndrome are heterozygous for Gly71Arg in the coding region of this gene. On the other hand, some patients with chronic hepatitis often show signs of unexpected hyperbilirubinemia. The aims of this study were to determine which of the genetic variations, TATA box genotype or codon 71 genotype, is most closely related to serum bilirubin concentrations, and whether the TATA box genotype has an effect on serum bilirubin concentrations in patients with hepatitis C-associated chronic liver diseases. In a sample of 300 individuals selected from among the general Japanese population, mean concentrations of total serum bilirubin differed significantly among TATA box genotypes, but not among codon 71 genotypes. Concentration of total serum bilirubin was significantly correlated with TATA box genotypes. In 211 patients with hepatitis C-associated chronic liver diseases, mean concentrations of total serum bilirubin also differed significantly among TATA box genotypes. In patients with chronic hepatitis C, concentration of total serum bilirubin was significantly correlated with TATA box genotypes. In summary, TATA box genotypes, but not codon 71 genotypes, are closely related to serum bilirubin concentrations. TATA box genotypes should therefore be considered when evaluating hepatic function by serum bilirubin concentrations in cases of hepatitis C-associated chronic liver diseases.

Effect of bilirubin UDP-glucuronosyltransferase 1 gene tata box on serum bilirubin concentrations in chronic liver injury

Effect of bilirubin UDP-glucuronosyltransferase 1 gene tata box on serum bilirubin concentrations in chronic liver injury

A new polymorphism in the APOE promoter associated with risk of developing Alzheimer's disease

The epsilon4 allele of the Apolipoprotein E gene (APOE), one of the main allele of APOE polymorphism, is a major risk factor for the development of Alzheimer's disease. However, several data suggest that genetic factors, within the APOE locus, may also modulate the risk associated with this polymorphism. We look for new mutations in the APOE promoter, susceptible to modify the risk associated with the APOE epsilon4 allele. We characterised a G-->T mutation at -186 bp of the APOE gene TATA box, named Th1/E47cs. This new polymorphism is located in a consensus sequence of a potential transcriptional (Th1/E47) factor binding site. We studied the impact of this new polymorphism with those of other markers of the APOE locus in a large case-control study and observed that Th1/E47cs modulated the influence of the APOE epsilon4 allele on the risk of Alzheimer's disease.

Cyclic AMP-responsive Expression of the Surfactant Protein-A Gene Is Mediated by Increased DNA Binding and Transcriptional Activity of Thyroid Transcription Factor-1

Surfactant protein (SP)-A gene transcription is stimulated by factors that increase cyclic AMP. In the present study, we observed that three thyroid transcription factor-1 (TTF-1) binding elements (TBEs) located within a 255 base pair region flanking the 5'-end of the baboon SP-A2 (bSP-A2) gene are required for maximal cyclic AMP induction of bSP-A2 promoter activity. We found that TTF-1 DNA binding activity was increased in nuclear extracts of pulmonary type II cells cultured in the presence of cyclic AMP. By contrast, the levels of immunoreactive TTF-1 protein were similar in nuclear extracts of control and cyclic AMP-treated type II cells. The incorporation of [32P]orthophosphate into immunoprecipitated TTF-1 protein also was markedly increased by cyclic AMP treatment. Moreover, exposure of nuclear extracts from cyclic AMP-treated type II cells either to potato acid phosphatase or alkaline phosphatase abolished the cyclic AMP-induced increase in TTF-1 DNA-binding activity. Interestingly, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), known to activate protein kinase C, also enhanced incorporation of [32P]orthophosphate into TTF-1 protein; however, the DNA binding activity of TTF-1 was decreased in nuclear extracts of TPA-treated type II cells. Expression vectors encoding TTF-1 and the catalytic subunit of protein kinase A (PKA-cat) were cotransfected into A549 lung adenocarcinoma cells together with an SPA:human growth hormone fusion gene (255 base pairs of 5'-flanking DNA from the baboon SP-A2 gene linked to human growth hormone, as reporter) containing TBEs, or with a reporter gene construct containing three tandem TBEs fused upstream of the bSP-A2 gene TATA box and the transcription initiation site. Coexpression of TTF-1 and PKA-cat increased fusion gene expression 3-4-fold as compared with expression of TTF-1 in the absence of PKA-cat. Moreover, the transcriptional activity of TTF-1 was suppressed by cotransfection of a dominant negative form of PKA regulatory subunit RIalpha. We suggest that a PKA-induced increase of TTF-1 phosphorylation and TBE binding activity mediates cyclic AMP-induced expression of the SP-A gene in lung type II cells.

Repeated sequences from the Arabidopsis thaliana genome function as enhancers in transgenic tobacco.

Sixteen segments of Arabidopsis thaliana DNA that function as enhancers in transgenic tobacco plants were isolated using the pROA97 enhancer cloning vehicle and library transformation of Nicotiana tabacum. The sequences were compared for AT content, homology, repeated motifs, and expression pattern in transgenic N. tabacum. The sequences were average with respect to the AT content of A. thaliana DNA. They could be placed into seven homology groups. Five of the sequences are single-copy sequences. The remaining eleven sequences represent two homology groups. Homology Group I contains seven sequences with minor differences. Homology Group II contains four sequences with minor differences. Two repeated motifs were identified (5'-CCTCT-3' and 5'-AAGGAT-3'). Both repeated motifs are found in other plant enhancers, and in the promoter region of the cauliflower mosaic virus 35S gene. In the 35S gene TATA region, the motifs can form two alternative stem-loop structures. The TATATAA sequence is located in the loop region of both stem-loop structures.

Repression of the alpha0 gene by ICP4 during a productive herpes simplex virus infection.

During a productive infection by herpes simplex virus type 1 (HSV-1), ICP4, the major regulatory protein encoded by the alpha4 gene, binds to its transcription initiation site and represses the accumulation of alpha4 RNA. Evidence suggests that the degree of repression by ICP4 is a function of the absolute distance of an ICP4 binding site 3' from a TATA box. However, repression of HSV-1 gene expression by ICP4 through binding sites located 5' of TATA boxes, as in the case of the alpha0 gene, has not been adequately addressed. To this end, recombinant alpha0 promoters with various arrays of ICP4 binding sites flanking the alpha0 TATA box were constructed and recombined into the HSV-1 genome. Our results demonstrate the following. (i) Destruction of the endogenous alphaO ICP4 binding site, located 5' of the TATA box, results in derepression of alpha0 protein and RNA accumulation in infected Vero cells. (ii) The degree of alpha0 derepression is equivalent to that reported for the alpha4 gene following destruction of the ICP4 binding site at the alpha4 mRNA cap site in HSV-1. (iii) Introduction of an ICP4 binding site at the alpha0 mRNA cap site represses the accumulation of alpha0 RNA greater than threefold relative to the wild type. (iv) Changes in the abundance of alpha0 protein and RNA in infected cells do not affect replication or growth of HSV-1 in tissue culture. Our findings are consistent with the conclusion that alpha0 transcription is repressed by ICP4. These results demonstrate that repression by ICP4 can occur through binding sites located 5' of virus gene TATA boxes in HSV-1. Thus, models addressing repression of HSV-1 gene expression by ICP4 should incorporate the role of binding sites located 5', as well as 3', of virus gene TATA boxes.

Molecular characterisation of the gonadotropin-releasing hormone receptor gene

The gonadotropin-releasing hormone (GnRH) receptor is a plasma membrane associated receptor and a member of the GTP-binding protein coupled receptor family. The interaction of GnRH and its receptor is a critical event in the endocrine control of reproduction. We have recently isolated the gene encoding the human pituitary GnRH receptor. The genomic clones obtained encompassed the full-length coding region and substantial portions of the 5′ and 3′ non-coding regions. Analysis of the clones revealed that the gene contains three exons and two introns which are distributed over 18.9 kb. Five putative promoters and transcription initiation sites have been identified in the 5′ end of the gene. Several consensus cis-acting regulatory sequences, such as sites for PEA-3, AP-1 and Pit-1, were identified within the 5′ flanking region. At the 3′ end of the gene, five classical polyadenylation signals were found scattered over a region of 800 bp. The mouse, sheep and rat GnRH receptor genes have also been characterised partially; these genes show similar structure and identical exon-intron splicing sites to the human GnRH receptor gene. However, the transcription start sites of the mouse and rat GnRH receptor genes are much closer to the translation start site when compared with the human GnRH receptor gene and multiple TATA boxes were not found in the promoter region of the mouse and rat GnRH receptor genes. On the other hand, initial characterisation of the ovine GnRH receptor gene indicates that, like the human gene, it contains multiple transcription initiation sites and far more 5′ untranslated region than the murine gene. Characterisation of the GnRH receptor gene provides the basis for further study of the complex mechanisms involved in the transcriptional regulation of GnRH receptor gene expression.

The symmetry of the yeast U6 RNA gene's TATA box and the orientation of the TATA-binding protein in yeast TFIIIB.

The central RNA polymerase III (Pol III) transcription factor TFIIIB is composed of the TATA-binding protein (TBP), Brf, a protein related to TFIIB, and the product of the newly cloned TFC5 gene. TFIIIB assembles autonomously on the upstream promoter of the yeast U6 snRNA (SNR6) gene in vitro, through the interaction of its TBP subunit with a consensus TATA box located at base pair -30. As both the DNA-binding domain of TBP and the U6 TATA box are nearly twofold symmetrical, we have examined how the binding polarity of TFIIIB is determined. We find that TFIIIB can bind to the U6 promoter in both directions, that TBP is unable to discern the natural polarity of the TATA element and that, as a consequence, the U6 TATA box is functionally symmetrical. A modest preference for TFIIIB binding in the natural direction of the U6 promoter is instead dictated by flanking DNA. Because the assembly of TFIIIB on the yeast U6 gene in vivo occurs via a TFIIIC-dependent mechanism, we investigated the influence of TFIIIC on the binding polarity of TFIIIB. TFIIIC places TFIIIB on the promoter in one direction only; thus, it is TFIIIC that primarily specifies the direction of transcription. Experiments using TFIIIB reconstituted with the altered DNA specificity mutant TBPm3 demonstrate that in the TFIIIB-U6 promoter complex, the carboxy-terminal repeat of TBP contacts the upstream half of the TATA box. This orientation of yeast TBP in Pol III promoter-bound TFIIIB is the same as in Pol II promoter-bound TFIID and in TBP-DNA complexes that have been analyzed by X-ray crystallography.

In vivo footprinting of the human IL-2 gene reveals a nuclear factor bound to the transcription start site in T cells.

The IL-2 gene is a T cell specific gene that is expressed early during the activation-specific T lymphocyte development program. Electrophoretic mobility shift assay (EMSA) and DNase I footprinting assays have defined DNA/protein interactions at the IL-2 promoter cis-elements in vitro. To determine if the trans-activators documented in T cell nuclear extracts actually bind the IL-2 promoter in vivo, ligation mediated PCR (LMPCR) genomic footprinting was performed on the IL-2 promoter in both activated and non-activated T cells and HL60 promyelocytes, which do not express the IL-2 gene. The in vivo footprints indicate that the IL-2 gene transcription start site and TATA sequence are protected in both activated and resting T cells, prior to the appearance of detectable IL-2 steady state message. The distal NF-AT and the NF kappa B sites are each footprinted and the Oct/OAP site contains hypersensitive residues in the unstimulated T lymphocytes. Additional residues are protected in each of these sites after T cell activation. The proximal NF-AT site (NF-IL-2B) and the AP-1 site at -150 are protected in activated Jurkat T lymphocytes, but these two sites are not protected in activated Jurkat lymphocytes stably transfected a gene construct containing multiple NFAT binding sites.

A nuclear protein is required for thyroid hormone receptor binding to an inhibitory half-site in the epidermal growth factor receptor promoter.

The epidermal growth factor (EGF) receptor (EGFR) promoter is negatively regulated by thyroid hormone and retinoic acid. This regulation can be mapped to a 36-basepair GC-rich region of the promoter (EGFR P/E) that functions autonomously as a promoter and an enhancer when placed in front of the thymidine kinase gene TATA element. Direct high affinity binding of the thyroid hormone receptor (T3R) to this element requires a nuclear protein. Through ion exchange chromatography and gel filtration of HeLa nuclear extract, this activity was identified as a protein of approximately 67 kilodaltons. This protein did not bind to DNA alone, but greatly augmented T3R binding to the EGFR P/E sequence in gel mobility shift and DNA precipitation assays. When combined with the T3R auxillary protein (TRAP), the T3R migrated as a larger complex on the DNA. Chemical cross-linking identified this complex as a heterodimer between T3R and TRAP. T3R-TRAP binds to a 7-basepair site in the EGFR P/E (GGGACTC) that has weak homology to a consensus thyroid response element half-site. Thus, on this element, T3R-TRAP heterodimers contact the DNA primarily on a single site that comprises an inhibitory thyroid response element.

Nuclear proteins and prolactin-induced annexin Icp35 gene transcription.

Annexin Icp35 is the major PRL-stimulated gene in the pigeon cropsac. The regulation of its promoter has been studied by in vitro assays for nuclear protein binding and transcription. Proteins present in nuclear extracts of PRL-stimulated, but not control, pigeon cropsac contained factors which specifically bound to sequences within 73 base pairs upstream of the cp35 transcription start point. The binding of some of the factors was localized to a region between -32 and -73 by gel-shift assays. Cell-free transcription was used to determine whether the cp35 promoter could be influenced by factors present in cropsac nuclear extract. HeLa cell nuclear extract transcribed the cp35 gene at a basal rate. Transcription of the cp35 gene, but not adml, was synergistically enhanced by nuclear extract from PRL-stimulated cropsac. Nuclear extract from unstimulated pigeon cropsacs did not stimulate either cp35 or adml transcription. A template from which sequences upstream of the cp35 gene TATA box were deleted was transcribed by HeLa cell extract but unaffected by any cropsac factors. These results demonstrate that PRL can cause the expression of one or more nuclear factors that bind to 5'-flanking DNA of cp35 and activate the gene's transcription.

Alpha and beta thyroid hormone receptors bind immediately adjacent to the rat growth hormone gene TATA box in a negatively hormone-responsive promoter region.

We report that alpha and beta type rat thyroid hormone receptors bind specifically and with high affinity to the 10-base pair sequence immediately 3' of the rat growth hormone TATA box (positions -25 to -16) in a region of the rat growth hormone promoter which can be negatively hormone responsive (nTRE). The receptors have approximately 7-fold lower affinity in vitro for the nTRE than for the thyroid hormone-responsive enhancer of the rat growth hormone gene (TRE). Proteins extracted with high salt concentration from rat pituitary cell nuclei enhance binding of the receptors to both the TRE and nTRE. A modification of the avidin-biotin complex DNA binding assay which enhances the sensitivity of the assay approximately 100-fold was used in these studies. The immediate proximity of a receptor binding site to the rat growth hormone TATA box suggests that direct interaction between receptor and TFIID (the TATA binding protein) mediates nTRE activity.

Functional dissection of an early Drosophila chorion gene promoter: expression throughout the follicular epithelium is under spatially composite regulation.

We have fused various DNA sequences located upstream of the Drosophila melanogaster s36 chorion gene TATA box to a heterologous basal promoter and reporter gene (hsp70/lacZ). The expression of these constructs, following P-element-mediated germline transformation, was examined in 144 independent lines by histological staining of dissected ovaries for beta-galactosidase activity. A short 84 bp segment of the proximal 5' flanking DNA was sufficient to confer a wild-type gene expression pattern, including temporal specificity for early choriogenic follicles. Surprisingly, initial expression was very localized at the anterior and posterior poles of the follicle. The downstream half of that DNA segment permitted expression at both poles, but especially at the anterior tip, while the upstream half only favored expression in the posterior pole; these results suggested the existence of multiple, spatially specific cis-regulatory elements. When the proximal 84 bp segment was placed 1.5 kb upstream of the basal promoter, beta-galactosidase activity was observed in an altered spatial pattern, indicating that the cis-regulatory element(s) that favor expression in the posterior half of the follicle are position independent, while the element(s) that favor expression elsewhere in the follicle are position sensitive. A distal regulatory segment containing redundant DNA element(s) specific for expression in the anterior pole was identified much further upstream of s36. Thus, the expression of this chorion gene throughout the follicular epithelium is actually composite, occurring in distinct spatial domains under the control of corresponding DNA elements.