CRE-like Sequence Research Articles

PepR1, a CcpA-like transcription regulator of Lactobacillus delbrueckii subsp. lactis.

The PepR1 protein from Lactobacillus delbrueckii subsp. lactis DSM 7290 shares extensive homology with catabolite-control proteins from various Gram-positive bacteria. Expression of the subcloned pepR1 gene allowed for partial complementation of a ccpA defect in Staphylococcus xylosus. The influence of PepR1 on transcription of the prolidase gene pepQ, which is located adjacent to pepR1, was examined by use of lacZ reporter gene fusions in Escherichia coli. PepR1 stimulated transcription initiation at the pepQ promoter about twofold, and this effect required the integrity of a 14 bp palindromic cre-like sequence located 74 nt upstream of pepQ. In gel-mobility-shift assays, PepR1 specifically interacted with the pepQ promoter region and also with DNA fragments covering the promoters of the pepX, pepl and brnQ genes of Lb. delbrueckii subsp. lactis, which encode two additional peptidases and a branched-chain amino acid transporter, respectively. cre-like elements were identified in each of these DNA fragments. Catabolite control of PepQ was demonstrated in Lb. delbrueckii subsp. lactis. During growth with lactose the enzyme activity was twofold higher than in the presence of glucose, and corresponding differences were also detected in the level of pepQ transcription.

Analysis of genomic regions involved in regulation of the rabbit surfactant protein A gene in transgenic mice.

The gene encoding surfactant protein (SP) A, a developmentally regulated pulmonary surfactant-associated protein, is expressed in a lung-specific manner, primarily in pulmonary type II cells. SP-A gene transcription in the rabbit fetal lung is increased by cAMP. To delineate the genomic regions involved in regulation of SP-A gene expression, lines of transgenic mice carrying fusion genes composed of various amounts of 5'-flanking DNA from the rabbit SP-A gene linked to the human growth hormone structural gene as a reporter were established. We found that as little as 378 bp of 5'-flanking DNA was sufficient to direct appropriate lung cell-selective and developmental regulation of transgene expression. The same region was also sufficient to mediate cAMP induction of transgene expression. Mutagenesis or deletion of either of two DNA elements, proximal binding element and a cAMP response element-like sequence, previously found to be crucial for cAMP induction of SP-A promoter activity in transfected type II cells, did not affect lung-selective or temporal regulation of expression of the transgene; however, overall levels of fusion gene expression were reduced compared with those of wild-type transgenes.

A Novel 14-Base-Pair Regulatory Element Is Essential for In Vivo Expression of Murine β4-Galactosyltransferase-I in Late Pachytene Spermatocytes and Round Spermatids

During murine spermatogenesis, beginning in late pachytene spermatocytes, the beta4-galactosyltransferase-I (beta4GalT-I) gene is transcribed from a male germ cell-specific start site. We had shown previously that a 796-bp genomic fragment that flanks the germ cell start site and contains two putative CRE (cyclic AMP-responsive element)-like motifs directs correct male germ cell expression of the beta-galactosidase reporter gene in late pachytene spermatocytes and round spermatids of transgenic mice (N. L. Shaper, A. Harduin-Lepers, and J. H. Shaper, J. Biol. Chem. 269:25165-25171, 1994). We now report that in vivo expression of beta4GalT-I in developing male germ cells requires an essential and previously undescribed 14-bp regulatory element (5'-GCCGGTTTCCTAGA-3') that is distinct from the two CRE-like sequences. This cis element is located 16 bp upstream of the germ cell-specific start site and binds a male germ cell protein that we have termed TASS-1 (transcriptional activator in late pachytene spermatocytes and round spermatids 1). The presence of the Ets signature binding motif 5'-GGAA-3' on the bottom strand of the TASS-1 sequence (underlined sequence) suggests that TASS-1 is a novel member of the Ets family of transcription factors. Additional transgenic analyses established that an 87-bp genomic fragment containing the TASS-1 regulatory element was sufficient for correct germ cell-specific expression of the beta-galactosidase reporter gene. Furthermore, when the TASS-1 motif was mutated by transversion, within the context of the original 796-bp fragment, transgene expression was reduced 12- to 35-fold in vivo.

Retinoic Acid Synergizes with Cyclic AMP to Enhance MMP-2 Basal Promoter Activity

Matrix metalloproteinase-2 (MMP-2) degrades basement membrane collagen and its abnormal expression is associated with the enhanced malignancy of metastasizing cancer cells. Retinoids and cyclic AMP analogs have been shown to affect MMP-2 production. Here we demonstrate that the expression of the human MMP-2 gene is enhanced by a synergistic action of retinoic acid (RA) and dibutyryl cyclic AMP (Bt2cAMP). RA also synergizes with Bt2cAMP in enhancing the basal promoter activity when the MMP-2 proximal promoter activity is induced by transient transfection and RA/Bt2cAMP treatment in human fibrosarcoma HT1080 cells. Deletions beyond −315 bp from the transcription initiation site drastically reduce the synergistic enhancement. A search for acis-element in the MMP-2 proximal promoter shows the presence of a CRE-like sequence (TGACGTCCC) at position −292 bp in the opposite strand. The CRE-like sequence makes complexes with two DNA binding proteins. The results demonstrate that the RA/Bt2cAMP-enhanced transcription of the MMP-2 gene is dependent on the general transcription machinery and suggest that the basal promoter may be a potential target for gene-specific activators.

CRITICAL INVOLVEMENT OF HUMAN T-CELL LEUKEMIA VIRUS TYPE 1 TAX PROTEIN IN IL-2-INDEPENDENT TRANSFORMATION OF T-CELLS

O-13 Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent of adult T-cell leukemia. Tax, the viral protein, is thought to be crucial in the development of the disease, since it is transforming in vitro and induces tumors in transgenic animals. We examined the effect of Tax activity on the growth of an interleukin (IL)-2-dependent T-cell line, CTLL-2. A stable expression of Tax in CTLL-2 converted cell growth from being IL-2-dependent to IL-2-independent. Tax stimulated the transcription through NF-κB and cAMP responsive element like sequence (CRE) in HTLV-1 promoter. Tax mutants segregating these two pathways suggested that the NF-κB pathway was essential for IL-2-independent growth of CTLL-2, while that of CRE was unnecessary. However, both pathways were necessary for another transformation-related activity (colony formation in soft agar) of CTLL-2/Tax. Our results show that Tax has at least two distinct activities on T-cells, and suggest that Tax plays a crucial role in IL-2-independent T-cell transformation induced by HTLV-1 in addition to its well known IL-2-dependent cell transformation.

Analysis of differentiation-induced expression mechanisms of thyrotropin receptor gene in adipocytes.

Rat adipose tissue, as well as differentiated 3T3-L1 cells, has been shown to express TSH receptor (TSHR) mRNA in amounts approaching those in the thyroid. We investigated the molecular mechanisms of TSHR gene expression in adipose cells. Primer extension and cloned cDNA sequences showed that transcription of the TSHR gene in rat adipose tissue was from multiple start sites clustered between -89 to -68 bp and almost identical to those in FRTL-5 thyroid cells. By transient expression analysis, we localized, between -146 and -90 bp, a positive regulatory element, the activity of which was markedly increased after the differentiation of 3T3-L1 cells. Deoxyribonuclease I protection showed that nuclear extracts from differentiated 3T3-L1 cells strongly protected two sequences, from -146 to -127 bp, including a cAMP response element-like sequence and from -112 to -106 bp containing a putative Ets-binding sequence. In differentiated 3T3-L1 cells, disruption or deletion of either sequence was found to result in the loss of enhancer activity, suggesting both elements may synergistically activate the TSHR promoter. Electrophoretic mobility shift analysis revealed the induction of new protein/DNA complexes formed either with the cAMP response element-like site or with putative Ets elements after the differentiation into adipocytes. In contrast, nuclear proteins, whose binding to DNA was diminished after the differentiation of 3T3-L1 cells, were found to interact with the site contiguous to the 5'-end of the putative Ets-binding sequence. Mutations of this binding site, which reduced the protein/DNA complex formation, increased TSHR promoter activity in undifferentiated cells. These observations suggested that differentiation-induced diminution of suppressor interactions may allow the enhancers to synergistically activate the transcription of TSHR gene in adipocytes.

Analysis of Differentiation-Induced Expression Mechanisms of Thyrotropin Receptor Gene in Adipocytes

Rat adipose tissue, as well as differentiated 3T3-L1 cells, has been shown to express TSH receptor (TSHR) mRNA in amounts approaching those in the thyroid. We investigated the molecular mechanisms of TSHR gene expression in adipose cells. Primer extension and cloned cDNA sequences showed that transcription of the TSHR gene in rat adipose tissue was from multiple start sites clustered between −89 to −68 bp and almost identical to those in FRTL-5 thyroid cells. By transient expression analysis, we localized, between −146 and −90 bp, a positive regulatory element, the activity of which was markedly increased after the differentiation of 3T3-L1 cells. Deoxyribonuclease I protection showed that nuclear extracts from differentiated 3T3-L1 cells strongly protected two sequences, from− 146 to −127 bp, including a cAMP response element-like sequence and from −112 to −106 bp containing a putative Ets-binding sequence. In differentiated 3T3-L1 cells, disruption or deletion of either sequence was found to result in the loss of enhancer activity, suggesting both elements may synergistically activate the TSHR promoter. Electrophoretic mobility shift analysis revealed the induction of new protein/DNA complexes formed either with the cAMP response element-like site or with putative Ets elements after the differentiation into adipocytes. In contrast, nuclear proteins, whose binding to DNA was diminished after the differentiation of 3T3-L1 cells, were found to interact with the site contiguous to the 5′-end of the putative Ets-binding sequence. Mutations of this binding site, which reduced the protein/DNA complex formation, increased TSHR promoter activity in undifferentiated cells. These observations suggested that differentiation-induced diminution of suppressor interactions may allow the enhancers to synergistically activate the transcription of TSHR gene in adipocytes.

Deoxyribonucleic acid-protein interactions and expression of the human testis-specific lactate dehydrogenase promoter: transcription factor Sp1 plays a major role.

The human testis-specific lactate dehydrogenase c gene (ldh-c) shows an exceptionally large window of expression throughout pre- and postmeiotic stages of the male germ cell lineage. In order to characterize the multiple stage-specific transcription factors necessary for ldh-c expression, we previously characterized the human ldh-c core promoter. Here, we used a combination of gel retardation assays and an in vitro transcription system derived from human tissues to better define the elements that govern ldh-c transcription. Three classes of transcriptional regulators were defined by these experiments. 1) The Sp1 transcription factor is a testis-"enriched" protein that is absent from most somatic tissues and that appears to play a major role in determining ldh-c expression in the testis. Highest levels of Sp1 during spermatogenesis correlate with maxima of ldh-c expression. 2) The testis-specific cAMP response element modulator (CREM) transcription factor binds a cAMP response element (CRE)-like sequence located at position -433. This transcriptional activator might contribute to postmeiotic transcription of ldh-c. 3) Factors present in tissues negative for ldh-c expression appear to bind both the CRE-like sequence and an adjacent hormone response element. The presence of this element could be involved in regulating ldh-c through the glucocorticoid/androgen pathways at the early stages of ldh-c expression.

Involvement of p38 Mitogen-activated Protein Kinase Signaling Pathway in the Rapid Induction of the 78-kDa Glucose-regulated Protein in 9L Rat Brain Tumor Cells

We have previously shown that treatment with okadaic acid (OA) followed by heat shock (HS) (termed OA --> HS treatment) leads to rapid transactivation of the 78-kDa glucose-regulated protein gene (grp78) in 9L rat brain tumor cells. A cAMP-responsive element-like (CRE-like, TGACGTGA) promoter sequence and a protein kinase A signaling pathway are involved in this induction, and activation of both CRE binding protein (CREB) and activating transcription factor-2 (ATF-2) is required in the above process. Herein, we report that transactivation of grp78, as well as phosphorylation/activation of ATF-2, can be completely annihilated by SB203580, a highly specific inhibitor of p38 mitogen-activated protein kinase (p38(MAPK)). Activation of p38(MAPK) by OA --> HS is also substantiated by its own phosphorylation as well as the phosphorylation and activation of MAPK activating protein kinase-2 in cells subjected to this treatment. The involvement of p38(MAPK) in the activation of ATF-2, which leads to the transactivation of rat grp78, is confirmed by electrophoretic mobility shift assay using a probe containing the CRE-like sequence as well as by transient transfection assays with a plasmid containing a 710-base pair stretch of the grp78 promoter. Together with our previous studies, these results led us to conclude that phosphorylation/activation of CREB upon OA --> HS treatment is mediated by cAMP-dependent protein kinase, whereas that of ATF-2 is mediated by p38(MAPK). The transcription factors may bind to each other to form heterodimers that in turn transactivate grp78 by binding to the CRE-like element. This suggests that distinct signaling pathways converge on CREB-ATF-2, where each subunit is individually activated by a specific class of protein kinases. This may allow modulation of grp78 transactivation by diverse external stimuli.

A CRE-like sequence that binds CREB and contributes to cAMP-dependent regulation of the proximal promoter of the human aromatase P450 ( CYP19) gene

The major physiological regulator of human aromatase P450 gene expression in the ovary is follicle stimulating hormone (FSH), which acts by increasing intracellular cAMP levels. This study describes the identification of an element in the aromatase proximal promoter that is critical for the full transcriptional response of this promoter to cAMP. The cAMP-responsive element (CRE)-like sequence (CLS) was originally identified by its sequence similarity to a palindromic CRE, from which it differs by the insertion of a single cytosine. Mutation of the CLS in the context of 278 bp of 5′-flanking DNA resulted in the loss of cAMP-induced reporter gene expression in transfected ovarian luteal cells. A cell line survey EMSA revealed that CLS binding factors are ubiquitously distributed, although the migration pattern of CLS-nuclear protein complexes varied among different nuclear extracts. An extended half-site for binding members of the basic-leucine zipper class of transcription factors was found to be responsible for ovarian luteal cell nuclear protein binding and cAMP-dependent transcriptional transactivation. Competition and supershift EMSAs revealed that the CLS-nuclear protein complexes that regulate cAMP-induced transcription were indistinguishable from homodimeric CREB bound to the CRE oligonucleotide, yet the interaction with the CLS was of lower affinity.

The 5'-Flanking Region of the Ovarian Promoter of the Bovine CYP19 Gene Contains a Deletion in a Cyclic Adenosine 3',5'-Monophosphate-Like Responsive Sequence

Conversion of C19 steroids to estrogens is catalyzed by aromatase P450 (P450arom; the product of the CYP19 gene). In the ovary, P450arom is expressed in granulosa cells of both human (h) and bovine (b) follicles. After the ovulatory surge of gonadotropins, however, P450arom expression is maintained only in the luteinized granulosa cells of the human ovary and is absent from the bovine corpus luteum. We compared the regulation of expression of the ovary-specific human CYP19 (hCYP19ov) and the bovine CYP19 (bCYP19ov) gene by cAMP (forskolin) and sought to determine whether the divergence in the expression of P450arom with the onset of luteinization could be explained by specific cis-acting elements present uniquely in the 5′-flanking DNA of the hCYP19ov or bCYP19ov gene. We, therefore, subcloned DNA encompassing the promoters and 5′-flanking regions of the hCYP19ov or bCYP19ov gene into a promoterless luciferase vector. These constructs were transfected into luteinized bovine granulosa cells or bovine luteal cells in primary culture. Neither cell type exhibits endogenous expression of bovine P450arom. After transfection, cells were treated with either vehicle or 25μ m forskolin. There was little or no increase in luciferase activity after forskolin treatment in cells transfected with any of the bCYP19ov constructs, whereas all of the corresponding hCYP19ov constructs (−693/−16 to −214/−16 bp) expressed reporter activity in the presence of forskolin. This dramatic difference between the activities of the constructs of the two species occurred despite the fact that there is an 88% sequence identity between the bovine and human promoters in the region between− 214 to −16 bp. One possible explanation for this variability may be that the bCYP19ov gene has a 1-bp deletion in a cAMP-response element-like sequence (CLS) present at −208 to −201 bp in the hCYP19ov gene that we have shown to be critical for cAMP-stimulated transcription of hCYP19ov in the ovary. When this region of the bCYP19ov promoter was mutated to the hCLS, a partial restoration in luciferase activity was observed after forskolin treatment. Therefore, these results suggest that another sequence in this −214 bp region of the bCYP19ov gene is also contributing to the lack of expression of P450arom after luteinization in the bovine ovary. This lack of expression of the bCYP19ov gene may be due to the presence of a repressive trans-acting factor expressed with the onset of luteinization of the bovine granulosa cell. These results further suggest that in the cow, elements upstream of those employed by the hCYP19ov gene may have been recruited to facilitate regulated expression of the bCYP19ov gene in the absence of a functional CLS.

The 5'-flanking region of the ovarian promoter of the bovine CYP19 gene contains a deletion in a cyclic adenosine 3',5'-monophosphate-like responsive sequence.

Conversion of C19 steroids to estrogens is catalyzed by aromatase P450 (P450arom; the product of the CYP19 gene). In the ovary, P450arom is expressed in granulosa cells of both human (h) and bovine (b) follicles. After the ovulatory surge of gonadotropins, however, P450arom expression is maintained only in the luteinized granulosa cells of the human ovary and is absent from the bovine corpus luteum. We compared the regulation of expression of the ovary-specific human CYP19 (hCYP19ov) and the bovine CYP19 (bCYP19ov) gene by cAMP (forskolin) and sought to determine whether the divergence in the expression of P450arom with the onset of luteinization could be explained by specific cis-acting elements present uniquely in the 5'-flanking DNA of the hCYP19ov or bCYP19ov gene. We, therefore, subcloned DNA encompassing the promoters and 5'-flanking regions of the hCYP19ov or bCYP19ov gene into a promoterless luciferase vector. These constructs were transfected into luteinized bovine granulosa cells or bovine luteal cells in primary culture. Neither cell type exhibits endogenous expression of bovine P450arom. After transfection, cells were treated with either vehicle or 25 microM forskolin. There was little or no increase in luciferase activity after forskolin treatment in cells transfected with any of the bCYP19ov constructs, whereas all of the corresponding hCYP19ov constructs (-693/-16 to -214/-16 bp) expressed reporter activity in the presence of forskolin. This dramatic difference between the activities of the constructs of the two species occurred despite the fact that there is an 88% sequence identity between the bovine and human promoters in the region between -214 to -16 bp. One possible explanation for this variability may be that the bCYP19ov gene has a 1-bp deletion in a cAMP-response element-like sequence (CLS) present at -208 to -201 bp in the hCYP19ov gene that we have shown to be critical for cAMP-stimulated transcription of hCYP19ov in the ovary. When this region of the bCYP19ov promoter was mutated to the hCLS, a partial restoration in luciferase activity was observed after forskolin treatment. Therefore, these results suggest that another sequence in this -214 bp region of the bCYP19ov gene is also contributing to the lack of expression of P450arom after luteinization in the bovine ovary. This lack of expression of the bCYP19ov gene may be due to the presence of a repressive trans-acting factor expressed with the onset of luteinization of the bovine granulosa cell. These results further suggest that in the cow, elements upstream of those employed by the hCYP19ov gene may have been recruited to facilitate regulated expression of the bCYP19ov gene in the absence of a functional CLS.

Regulation of Major Histocompatibility Complex Class I Gene Expression in Thyroid Cells: ROLE OF THE cAMP RESPONSE ELEMENT-LIKE SEQUENCE

The major histocompatibility complex (MHC) class I gene cAMP response element (CRE)-like site, -107 to -100 base pairs, is a critical component of a previously unrecognized silencer, -127 to -90 bp, important for thyrotropin (TSH)/cAMP-mediated repression in thyrocytes. TSH/cAMP induced-silencer activity is associated with the formation of novel complexes with the 38-base pair silencer, whose appearance requires the CRE and involves ubiquitous and thyroid-specific proteins as follows: the CRE-binding protein, a Y-box protein termed thyrotropin receptor (TSHR) suppressor element protein-1 (TSEP-1); thyroid transcription factor-1 (TTF-1); and Pax-8. TTF-1 is an enhancer of class I promoter activity; Pax-8 and TSEP-1 are suppressors. TSH/cAMP decreases TTF-1 complex formation with the silencer, thereby decreasing maximal class I expression; TSH/cAMP enhance TSEP-1 and Pax-8 complex formation in association with their repressive actions. Oligonucleotides that bind TSEP-1, not Pax-8, prevent formation of the TSH/cAMP-induced complexes associated with TSH-induced class I suppression, i.e. TSEP-1 appears to be the dominant repressor factor associated with TSH/cAMP-decreased class I activity and formation of the novel complexes. TSEP-1, TTF-1, and/or Pax-8 are involved in TSH/cAMP-induced negative regulation of the TSH receptor gene in thyrocytes, suppression of MHC class II, and up-regulation of thyroglobulin. TSH/cAMP coordinate regulation of common transcription factors may, therefore, be the basis for self-tolerance and the absence of autoimmunity in the face of TSHR-mediated increases in gene products that are important for thyroid growth and function but are able to act as autoantigens.

Structural organization and characterization of the promoter region of the rat gonadotropin-releasing hormone receptor gene

The gene encoding the rat gonadotropin-releasing hormone (GnRH) receptor was isolated, and its structural organization and promoter region were characterized. The gene was found to consist of three exons that encode the receptor protein, and spanned about 20 kb. Of two genomic clones analyzed, one contained the 5′-untranslated region and the first exon, and the other contained the second and third exons. The sizes of the first, second, and third exons are 625, 217, and 1476 nt, respectively. The first intron is at least 12 kb in length and is located between nucleotides 522 and 523 of the cDNA reading frame, in the middle of the fourth transmembrane domain. The second intron is about 2.5 kb and is also located in the reading frame between nucleotides 739 and 740, separating the fifth and sixth transmembrane domains. Genomic blots in combination with cloning and sequencing suggested that a single GnRH receptor gene is present in the rat genome. Primer extension indicated that the transcription start site is located 103 nt upstream of the translational start codon. A putative TATA box is positioned 23 nt in front of the transcription initiation site. The 1.8 kb 5′ flanking sequence contains an SF-1 site, an AP-1 site, CCAAT sequences, a Pit-1 binding site, and a potential CRE-like sequence. To evaluate promoter activity, the 1.8 kb and two 5′ deleted fragments of 1.2 and 0.6 kb were fused to the luciferase reporter gene and transiently expressed in immortalized pituitary gonadotrophs ( αT3-1 cells) and hypothalamic neurons (GT1-7 cells), and in nonpituitary (COS-7) cells. Luciferase gene expression was significantly increased by all three fragments in pituitary and hypothalamic cells, but not in COS-7 cells. The promoter activity of the 1.2 kb fragment was higher than that of the other fragments. Forskolin and cAMP analogs increased luciferase gene expression in both αT3-1 and GT1-7 cells, but activation of protein kinase C by phorbol myristate acetate had no effect. These studies indicate that positive and negative regulatory elements are present within the 1.8 kb 5′ flanking sequence of the GnRH receptor. Knowledge of the genomic organization and analysis of the promoter region of the rat GnRH receptor gene will facilitate the elucidation of its transcriptional control in pituitary gonadotrophs and hypothalamic neurons.

Phosphorylation and high level expression of Fra-2 in v-src transformed cells: a pathway of activation of endogenous AP-1.

Chicken embryo fibroblasts (CEF) transformed with v-src were previously reported to revert to normal phenotype after the introduction of dominant-negative mutants of Fos or Jun, indicating that endogenous AP-1 activity is essential for the cellular transformation. The major changes in the expression levels of fos and jun family genes induced by v-src were the elevation of fra-2 and c-jun transcripts. We show here that extensive phosphorylation of the AP-1 component Fra-2 is a major qualitative change in v-src transformed CEF and that several Ser and Thr residues in a C-terminal region of Fra-2 (amino acids 266-323) are phosphorylated specifically. The induced kinase activity was detected at the position of 42 kDa by in gel kinase assay using the Fra-2 C-terminal region as a substrate, and it was identified as chicken ERK2. JNK1 and JNK2, other members of the MAP kinase family, were not significantly activated in v-src transformed CEF and Fra-2 was not a good substrate for JNKs. fra-2 promoter analysis indicated that this promoter activity is elevated in v-src transformed CEF via two AP-1 binding sites and CRE-like sequence. We propose that phosphorylation of Fra-2 by ERK2 converts it from an inefficient transcriptional activator to an active one and further that fra-2 expression is autoregulated in response to the phosphorylation status of its gene product.

Isolation and Characterization of the Rat Corticotropin-Releasing Hormone (CRH)-Binding Protein Gene: Transcriptional Regulation by Cyclic Adenosine Monophosphate and CRH1

The CRH-binding protein (CRH-BP) antagonizes the ACTH-releasing activity of the neuropeptide CRH in vitro. However, the function of CRH-BP in vivo and the molecular mechanisms that regulate CRH-BP expression are not well understood. In this study, the rat CRH-BP gene was characterized, and CRH-BP promoter sequences were identified. The rat CRH-BP gene spans almost 12 kilobases and contains 7 exons. Ribonuclease protection experiments indicate that transcription of the CRH-BP gene initiates at multiple sites in rat cerebral cortex. Transfection experiments with CRH-BP-reporter constructs, containing 88-3500 bp 5' flanking and 66 bp 5' untranslated DNA from the rat CRH-BP gene, demonstrate basal promoter activity in multiple cell lines. CRH-BP-reporter constructs also demonstrate positive regulation of promoter activity by cAMP in a variety of cell lines and by CRH in cells expressing the CRH receptor. The DNA sequences between -341 and -88 bp, including the cAMP response element-like sequence at -127 bp, are required for maximal cAMP and CRH regulation of CRH-BP promoter activity. These studies suggest that CRH-BP transcription in vivo may be positively regulated by cAMP and CRH.

Regulation of aromatase expression in the ovary and placenta: a comparison between two species

The conversion of C19 steroids to estrogens is catalysed by aromatase P450 (P450arom; product of the CYP19 gene). Tissue sites of expression include the gonads and brain; however, in a small subset of mammals, P450arom is also expressed in the placenta. In humans, gonadal expression employs a promoter proximal to the start site of translation, whereas expression in the placenta relies on a promoter which is distal to this site. We characterized the bovine CYP19 gene in both the ovary (OV) and placenta (PL), to determine if this method of regulation of tissue-specific expression is employed in other species. Both bovine and human species express P450arom in these tissues, however, the pattern of expression is very different. In both humans and cattle, P450arom is expressed in the granulosa cells of the ovary, however, after ovulation only the luteinized granulosa cells of the human continue to express P450arom. There is a high degree of sequence identity (>70%) shared between humans and cattle in the OV-specific 5′-flanking DNA, whereas little identity (<40%) was found between humans and cattle in the PL-specific DNA. Promoters and 5′-flanking regions of human and bovine CYP19 genes were subcloned into a luciferase (LUC) vector. Bovine PL-specific constructs transfected into JEG-3 human choriocarcinoma cells failed to express LUC activity. When OV-specific constructs were transfected into luteinized bovine granulosa cells, there was no change in LUC activity in cells transfected with the bovine constructs after treatment with forskolin, whereas all of the corresponding human constructs expressed LUC activity. The bovine 5′-flanking DNA lacks a cAMP-responsive element-like sequence (CLS) critical for cAMP-stimulated transcription of P450arom in the human ovary. With the absence of this CLS sequence in the bovine gene, there appears to be little enhancement of transcription by cAMP in the portion of the 5′-flanking region studied so far. When the analogous region of the bovine promoter was mutated to the human CLS, only a partial restoration of LUC activity was observed. An additional element therefore appears to be important in preventing the full expression of the bovine CYP19 gene in luteinized granulosa cells.

Evidence that Functional Interactions of CREB and SF-1 Mediate Hormone Regulated Expression of the Aromatase Gene in Granulosa Cells and Constitutive Expression in R2C Cells

The proximal promoter of the rat aromatase CYP19 gene contains two functional domains that can confer hormone/cAMP inducibility in primary cultures of rat granulosa cells and constitutive expression in R2C Leydig cells. Region A contains a hexameric sequence that binds steroidogenic factor-1 (SF-1). Region B contains a CRE-like sequence that binds CREB and two other factors, X and Y. To determine if CRE binding factors X and Y had overlapping functions with CREB, and to determine if the CREB and SF-1 binding sites exhibited functional interactions in the context of the intact promoter, mutations within the CRE and hexameric SF-1 binding site were generated. Mutations within the CRE showed that CREB but not factors X and Y mediated cAMP-dependent activity of chimeric transgenes in primary granulosa cell cultures. Granulosa cells transfected with constructs that bound CREB but not SF-1 (or the converse) resulted in a loss of approximately 50% cAMP-dependent CAT activity. Transgenes that did not bind CREB or SF-1 exhibited no cAMP-dependent CAT activity. When these same constructs where transfected into R2C Leydig cells, mutation of either the CREB or SF-1 binding sites resulted in a greater than 90% loss of CAT activity. Western blot and immunocytochemistry analyses revealed that the amount of phosphorylated CREB increased in response to hormone/cAMP in granulosa cells and was high in R2C Leydig cells, coinciding with expression of the transgenes and endogenous aromatase mRNA in each cell type. Therefore, in both cell types the aromatase promoter is dependent upon a functional CRE and the presence of phosphoCREB. The CREB and SF-1 binding sites interact in an additive manner to mediate cAMP transactivation in granulosa cells, whereas they interact synergistically to confer high basal transactivation in R2C Leydig cells. Taken together, the results indicated that the molecular mechanisms or pathways that activate CREB, SF-1 or their interaction are different in granulosa cells and R2C cells.

Evidence that functional interactions of CREB and SF-1 mediate hormone regulated expression of the aromatase gene in granulosa cells and constitutive expression in R2C cells

The proximal promoter of the rat aromatase CYP19 gene contains two functional domains that can confer hormone/cAMP inducibility in primary cultures of rat granulosa cells and constitutive expression in R2C Leydig cells. Region A contains a hexameric sequence that binds steroidogenic factor-1 (SF-1). Region B contains a CRE-like sequence that binds CREB and two other factors, X and Y. To determine if CRE binding factors X and Y had overlapping functions with CREB, and to determine if the CREB and SF-1 binding sites exhibited functional interactions in the context of the intact promoter, mutations within the CRE and hexameric SF-1 binding site were generated. Mutations within the CRE showed that CREB but not factors X and Y mediated cAMP-dependent activity of chimeric transgenes in primary granulosa cell cultures. Granulosa cells transfected with constructs that bound CREB but not SF-1 (or the converse) resulted in a loss of approximately 50% cAMP-dependent CAT activity. Transgenes that did not bind CREB or SF-1 exhibited no cAMP-dependent CAT activity. When these same constructs where transfected into R2C Leydig cells, mutation of either the CREB or SF-1 binding sites resulted in a greater than 90% loss of CAT activity. Western blot and immunocytochemistry analyses revealed that the amount of phosphorylated CREB increased in response to hormone/cAMP in granulosa cells and was high in R2C Leydig cells, coinciding with expression of the transgenes and endogenous aromatase mRNA in each cell type. Therefore, in both cell types the aromatase promoter is dependent upon a functional CRE and the presence of phosphoCREB. The CREB and SF-1 binding sites interact in an additive manner to mediate cAMP transactivation in granulosa cells, whereas they interact synergistically to confer high basal transactivation in R2C Leydig cells. Taken together, the results indicated that the molecular mechanisms or pathways that activate CREB, SF-1 or their interaction are different in granulosa cells and R2C cells.

Genomic Structure and Promoter Characterization of the Human ACTH Receptor Gene

One kb of the 5′-flanking region of the human ACTH receptor gene was isolated and partially characterized. Transient transfections with hGH reporter confirmed the promoter activity in Y1 cells. Putative elements for transcription factors involved in regulation were noted in the sequence. The promoter activity of some of the constructs is responsive to a treatment by forskolin, due to the presence of several CRE-like sequences.