CCAAT Element Research Articles

SpSoxB1 serves an essential architectural function in the promoter SpAN, a tolloid/BMP1-related gene.

Transcription of SpAN, which encodes a secreted protease related to tolloid and BMP 1, is differentially regulated along the animal-vegetal axis of the sea urchin embryo by a maternally initiated mechanism. Regulatory sites that bind SpSoxB1 and CBF (CCAAT binding factor) are essential for strong transcriptional activity because mutations of these elements reduce promoter activity in vivo 20- and 10-fold, respectively. Here we show that multimerized SpSoxB1 elements cannot activate transcription from the SpAN basal promoter in vivo. However, like other factors containing HMG-class DNA binding domains, SpSoxB1 does induce strong bending of DNA. The CBF binding site lies abnormally far from the transcriptional start site at -200 bp. We show that the SpSoxB1 site is not required if the CCAAT element is moved 100 bp closer to the transcriptional start site, replacing the SpSoxB1 site. This supports a model in which the bending of SpAN promoter DNA by SpSoxB1 facilitates interactions between factors binding to upstream and downstream regulatory elements.

Analysis of Basal Regulatory Elements in the 25-Hydroxyvitamin D3 1α-Hydroxylase Gene Promoter

5′-Deletion analysis of the 1.7-kb mouse 1α-hydroxylase gene promoter reveals that the minimal promoter region for basal activity is −85/+22 and requires a functional CCAAT element. Mutational analysis also demonstrates that deletion of the internal promoter region from nucleotides −1125 to −86 leads to a 25- to 30-fold increase in basal promoter activity. The increased activity is not the result of positional effects, but is caused in part by the removal of an AC repeat. Further analysis of the promoter revealed an enhancer element localizes to an upstream region −1385 to −1125, which contains three consensus AP-1 sites. Deletion of the most proximal AP-1 site causes a 60% loss of enhancer activity. The data suggest the presence of the AC repeat prevents the full potential activation of the 1α-hydroxylase promoter by factors binding to AP-1 sites.

The Aspergillus nidulans multimeric CCAAT binding complex AnCF is negatively autoregulated via its hapB subunit gene

Cis-acting CCAAT elements are frequently found in eukaryotic promoter regions. Many of them are bound by conserved multimeric complexes. In the fungus Aspergillus nidulans the respective complex was designated AnCF ( A. nidulans CCAAT binding factor). AnCF is composed of at least three subunits designated HapB, HapC and HapE. Here, we show that the promoter regions of the hapB genes in both A. nidulans and Aspergillus oryzae contain two inversely oriented, conserved CCAAT boxes (box α and box β). Electrophoretic mobility shift assays (EMSAs) using both nuclear extracts and the purified, reconstituted AnCF complex indicated that AnCF binding in vitro to these boxes occurs in a non-mutually exclusive manner. Western and Northern blot analyses showed that steady-state levels of HapB protein as well as hapB mRNA were elevated in hapC and hapE deletion mutants, suggesting a repressing effect of AnCF on hapB expression. Consistently, in a hapB deletion background the hapB-lacZ expression level was elevated compared with the expression in the wild-type. This was further supported by overexpression of hapB using an inducible alcA-hapB construct. Induction of alcA-hapB expression strongly repressed the expression of a hapB-lacZ gene fusion. However, mutagenesis of box β led to a fivefold reduced expression of a hapB-lacZ gene fusion compared with the expression derived from a wild-type hapB-lacZ fusion. These results indicate that (i) box β is an important positive cis-acting element in hapB regulation, (ii) AnCF does not represent the corresponding positive trans-acting factor and (iii) that AnCF is involved in repression of hapB.

Transcriptional activation of the small GTPase gene rhoB by genotoxic stress is regulated via a CCAAT element.

The gene encoding the Ras-related GTPase RhoB-specific is immediate-early inducible by genotoxic treatments. Regulation of transcriptional activation of rhoB is still unclear. Here we show that cells lacking either p53 or c-Fos are not different from wild-type cells with respect to the level of rhoB induction upon UV irradiation, indicating that these transcription factors are not crucial for stimulation of rhoB mRNA expression. Extracts from UV-irradiated and non-irradiated cells revealed similar DNA-binding activities to a 0.17 kb rhoB promoter fragment harboring the functional element(s) necessary for stimulation of rhoB by UV light. By means of immunoprecipitation we found that an ATF-2-specific antibody co-precipitates the (32)P-labeled 0.17 kb rhoB fragment, whereas an anti-AP1 antibody did not. Since no consensus sequence for binding of ATF-2 is present within the rhoB promoter, ATF-2 is likely to be associated with another factor that binds to the minimal promoter. Deletion analysis and site-directed mutagenesis of the 0.17 kb rhoB fragment revealed a CCAAT box to be an essential requirement for stimulation of rhoB by UV light and methyl methanesulfonate. Moreover, immunoprecipitation experiments showed that the CCAAT-binding factor NF-YA is complexed with ATF-2. Overall, the data strongly indicate that transcriptional activation of the rhoB gene by genotoxic stress is regulated via a CCAAT box and that interaction of CCAAT-binding factor and ATF-2 triggers the stress-inducible expression of rhoB.

Role of an inverted CCAAT element in human topoisomerase IIalpha gene expression in ICRF-187-sensitive and -resistant CEM leukemic cells.

DNA topoisomerase (topo) IIalpha gene expression or activity is altered in tumor cells selected for resistance to inhibitors of topoII. To better understand the mechanisms by which topoIIalpha expression levels are modulated, we examined topoIIalpha transcriptional regulation in ICRF-187-sensitive and ICRF-187-resistant human leukemic cell lines that express an increased amount of topoIIalpha protein and mRNA. Transient transfections of luciferase reporter plasmids containing either the full-length human topoIIalpha promoter or fragments of it revealed that topoIIalpha transcriptional activity was significantly increased in the drug-resistant CEM/ICRF-8 cells, compared with CEM cells. Specifically, the transcriptional activity of the full-length topoIIalpha promoter (nucleotides -557 to +90) was doubled in CEM/ICRF-8 compared with CEM cells. Serial deletion of the topoIIalpha promoter permitted localization of the region responsible for its up-regulation in the drug-resistant cells between nucleotides -557 and -162, which includes the last three inverted CCAAT elements (ICE) 3 to 5. Note that construction of a point mutation in ICE3 resulted in a significant increase in transcriptional activity of the topoIIalpha promoter in the drug-sensitive CEM cells. In addition, by electrophoretic mobility shift assay, ICE3 was recognized by a protein complex containing NF-YB that was present at reduced levels in the topoIIalpha-overexpressing CEM/ICRF-8 extracts, suggesting that ICE3 plays a negative regulatory role in human topoIIalpha gene expression. This is the first study to show that topoIIalpha transcriptional up-regulation in ICRF-187-resistant cells is mediated in part by altered regulation of the third inverted CCAAT box in the topoIIalpha promoter.

The FIRE3-mediated sterol response of the FAS promoter requires NF-Y/CBF as a coactivator.

The transcription of the fatty acid synthase (FAS) gene is regulated by the sterol status of the cell via cleavage of the sterol regulatory element-binding protein (SREBP). When human HepG2 hepatoma cells were cotransfected with an expression plasmid for mature SREBP-1a together with FAS promoter/reporter constructs significant increases in reporter activity were observed. Deletion analysis of the FAS promoter between -151 and -52 relative to the transcription start site pinpoint two cis-elements important in sterol regulation of the FAS gene. One element, FIRE3, between -71 and -52 can bind in vitro translated and transcribed SREBP-1a whereas the other element, the inverted CCAAT element ICE(-97/-92), binds the trimeric transcription factor NF-Y/CBF as shown with rat liver extract and reconstituted, recombinant NF-Y. The results clearly show that the coactivator for SREBP-1a in this cell line is NF-Y. This finding was confirmed by using a dominant negative form of NF-YA, NF-YAm29, which interferes with the effect of ectopically expressed SREBP-1a on FAS reporter activity.

Functional Interaction of NF-Y and Sp1 Is Required for Type A Natriuretic Peptide Receptor Gene Transcription

The vasorelaxant and anti-mitogenic activities of the atrial and brain natriuretic peptides depend upon their binding to the type A natriuretic peptide receptor (NPR-A) expressed on the surface of vascular cells. Intervention strategies aimed at controlling NPR-A expression are limited by the paucity of studies in this area. Here we identify a sequence CCAAT between -141 and -137 of the NPR-A promoter that, when mutated, reduces promoter activity by 90% in rat aortic smooth muscle (RASM) cells. Protein/DNA cross-linking and immunoperturbation of electrophoretically shifted complexes formed between RASM nuclear extracts and an oligonucleotide surrounding the CCAAT sequence indicates that the heterotrimeric transcription factor NF-Y binds specifically to the wild-type, but not mutated, CCAAT element. Cotransfection of a dominant negative mutant of the NF-YA subunit results in a concentration-dependent decrease in the activity of the NPR-A promoter in RASM cells confirming that endogenous NF-Y is an activator of the promoter. Mutation of the CCAAT element, in conjunction with mutation of all three Sp1 sites previously shown to be involved in NPR-A promoter regulation, virtually eliminates NPR-A promoter activity in RASM cells. Coexpression of all three NF-Y subunits together with Sp1 in Drosophila cells deficient in these factors indicates that NF-Y and Sp1 act synergistically to reconstitute NPR-A promoter activity. A direct physical association between NF-Y and Sp1 can be demonstrated both in vitro by glutathione S-transferase pull-down assay and in the intact cell by coimmunoprecipitation and functional studies. Together, these studies show that NPR-A promoter activity is dominantly regulated through functional, and possibly physical, interactions of NF-Y and Sp1.

The NFY Transcription Factor Mediates Induction of the von Willebrand Factor Promoter by Irradiation

Ionizing irradiation in patients is proposed to cause thrombus formation. An increase in von Willebrand factor secretion in response to irradiation is a major contributing factor to thrombus formation. We have previously reported that the increased VWF secretion in response to irradiation is mediated at the transcriptional level. The VWF core promoter fragment (sequences -90 to +22) was shown to contain the necessary cis-acting element(s) to mediate the irradiation response of the VWF gene. Here we report that a CCAAT element in the VWF promoter is the cis-acting element necessary for irradiation induction and that the NFY transcription factor interacts with this element. These analyses demonstrate that inhibition of NFY's interaction with the CCAAT element abolishes the irradiation induction of the VWF promoter. These results provide a novel role for NFY and add this factor to the small list of irradiation-responsive transcription factors. Coimmunoprecipitation experiments demonstrated that NFY is associated with the histone acetylase P/CAF in vivo and that irradiation resulted in an increased association of NFY with coactivator P/CAF. We propose that irradiation induction of the VWF promoter involves a mechanism resulting in increased recruitment of the coactivator P/CAF to the promoter via the NFY transcription factor.

Complex Structure and Regulation of Expression of the Rat Gene for Inward Rectifier Potassium Channel Kir7.1

Genomic organization of the rat inward rectifier K(+) channel Kir7.1 was determined in an attempt to clarify how multiple species of its mRNA are generated in a tissue-specific manner and how its expression is regulated. The rat Kir7.1 gene spans >40 kilobases (kb) and consists of eight exons; the first four exons encode the 5'-untranslated region that is unusually long ( approximately 3 kb). The coding region is located in exons 5 and 6. In the testis, exon 4 is processed as four exons (4a-4d), whereas it is recognized as a single exon in the small intestine. The three major species of rat Kir7.1 mRNA (1.4, 2.2, and 3.2 kb) were found to arise from alternative usage of the two promoters and polyadenylation signals and by alternative splicing of the 5'-noncoding exons. The splicing pattern of the 5'-noncoding exons is quite complex and highly tissue-specific, suggesting that complex mechanisms may operate to regulate the Kir7.1 expression. Deletion and mutational analysis of the promoter activity indicated that the rat Kir7.1 gene is regulated by cAMP through a CCAAT element. The cAMP induction was also demonstrated using the rat follicular cell line FRTL-5 endogenously expressing Kir7.1.

Isolation and characterization of the human A-myb promoter: regulation by NF-Y and Sp1.

The A-myb transcription factor shows a restricted tissue distribution and is cell cycle regulated. Furthermore its deregulation has profound effects on the growth and/or differentiation of the cells in which it is normally expressed. We have therefore characterized its promoter. A 12 kb genomic clone was isolated that comprises the first exon, part of the first intron as well as upstream regulatory sequences. Multiple transcription start sites have been identified which operate in both B lymphocytes and epithelial cells and the upsteam region was shown to have promoter, activity. The boundaries of the minimal promoter region (-183-14), of a positive upstream (-538-183) and a negative downstream regulatory region (NRE) (+83+374) have been defined. The NRE is promoter- and orientation-independent but position specific. The A-myb minimal promoter is GC-rich, does not contain any TATA box but has a functional CCAAT box. The CCAAT box and minimal promoter is highly conserved in the corresponding murine sequence. The CCAAT box efficiently binds the NF-Y complex and its mutation decreases basal promoter activity by 50%. Two Sp1 binding sites are present upstream from the CCAAT box which can bind Spl and contribute to A-myb promoter activity by 70 and 30%, respectively. The two Sp1 sites and CCAAT box together contribute to over 80% of A-myb basal promoter activity and are therefore the major regulatory elements. Finally, we show that the promoter is cell cycle regulated and that the SP1 and CCAAT elements are required for S phase induction.

Erratum: analysis of DNA elements that modulate myosin VIIa expression in humans.

Usher syndromeIb (USH1B), an autosomal recessive disorder caused by mutations in myosin VIIa (MYO7A), is characterized by congenital profound hearing loss, vestibular abnormalities and retinitis pigmentosa. Promoter elements in the 5 kb upstream of the translation start were identified using adult retinal pigment epithelium cells (ARPE-19) as a model system. A 160 bp minimal promoter within the first intron was active in ARPE-19 cells, but not in HeLa cells that do not express MYO7A. A 100 bp sequence, 5' of the first exon, and repeated with 90% homology within the first intron, appeared to modulate expression in both cell lines. Segments containing these elements were screened by heteroduplex analysis. No heteroduplexes were detected in the minimal promoter, suggesting that this sequence is conserved. A -2568 A>T transversion in the 5' 100 bp repeat, eliminating a CCAAT element, was found only in USH1B patients. However, in all 5 families, -2568 A>T was in cis with the same missense mutation in the myosin VIIa tail (Arg1240Gln), and 4 of the 5 families were Dutch. These observations suggest either 1) linkage disequilibrium or 2)that a combination of a promoter mutation with a less active myosin VIIa protein results in USH1B.

Partial characterization of the CCAAT box in the promoter of the hIGFBP-1 gene: interaction with negatively acting transcription factors in decidualized human endometrial stromal cells

The CCAAT cis-element and its adjacent DNA sequence (−82 to −52 bp) in the human insulin-like growth factor binding protein-1 gene (IGFBP-1) promoter are active in both decidualized human endometrial stromal cells and HepG2 cells. In HepG2 cells, CCAAT activity is mediated by interacting with hepatocyte nuclear factor, HNF-1. In endometrial cells, this region is protected by the nuclear extracts of endometrial decidual cells, however, the transactivator which interacts with the region has not been identified. This study was carried out to characterize and identify the stromal/decidual nuclear proteins that interact with the IGFBP-1 CCAAT motif. Gel shift analysis showed that the CCAAT motif (−82 to −52 bp) formed three specific complexes (CI, CII, and CIII) by extracts from human endometrial decidual or stromal cells. The intensity of CIII formed by the nuclear extracts of decidual cells was less compared to that formed by stromal cells whereas CI/CII was found to be opposite. To evaluate the transcription factors that bind to this region, a number of known CCAAT binding proteins were tested. Among them, the CCAAT binding proteins NF-Y (α2(1) collagen promoter CCAAT binding protein) and CBF (hsp70 promoter CCAAT binding protein), were characterized by the gel shift assay. The NF-Y consensus binding sequence (the α2(1) collagen promoter) and NF-YA,B antibody abolished or shifted CIII. Although the CBF consensus binding sequence (the hsp70 promoter) eliminated all three complexes, the antibody to CBF had no effect on all three complexes. The nuclear extracts of the endometrial stromal/decidual cells did not form a band corresponding to the HNF-1/CCAAT complex. These results indicate that the CCAAT motif binds to NF-Y and the CI/CII binding protein (remains to be identified) but not HNF-1 in endometrium. Systematic mutation in the CCAAT motif showed that NF-Y(CIII binding protein) bound to the 12 bp sequence GGCGCTG CCAAT (−79 to −68 bp) and the CI/CII binding protein bound to 9 bp, TG CCAAT CA(−74 to −66 bp). These findings indicate that the CCAAT motif is a composite element. The CCAAT mediated function was analyzed in decidualized endometrial stromal cells. Mutations in the CCAAT motif increased the promoter activity. The maximum activity was found in mutants which abolished the NF-Y complex. The CCAAT core sequence mutants in which both CIII and CI/CII were abolished, also increased the promoter activity. Results indicated that NF-Y and the CI/CII binding protein, yet to be identified, interact with the composite CCAAT element in the IGFBP-1 promoter to repress the promoter activity in endometrial decidual cells.

Characterization of Proximal Transcription Regulatory Elements in the Rat Phospholamban Promoter

C. F. McTiernan, B. H. Lemster, C. S. Frye, D. C. Johns and A. M. Feldman. Characterization of Proximal Transcription Regulatory Elements in the Rat Phospholamban Promoter. Journal of Molecular and Cellular Cardiology (1999)31 , 2137–2153. Phospholamban is a major regulator of cardiac diastole, with alterations in expression associated with modified cardiac relaxation. To study transcriptional regulation of phospholamban expression, we made reporter constructs that expressed luciferase under control of putative promoter sequences from the rat phospholamban gene. When transfected into neonatal rat cardiomyocytes, constructs containing at least 159 nucleotides preceding the transcription start site were equally active, while truncation to −66/+64 removed all promoter activity. Constructs were more active in cardiomyocytes than in HeLa cells (which do not express phospholamban), but did not show absolute cell-type specificity of expression. Addition of sequences upstream to −4032, all of the intron (7.4 kb), or 3′UTR sequences (0.8 kb) did not enhance cell-specific expression. To focus on the basal promoter region (−159/−66), a series of deletion constructs were made that identified a novel 35 bp region (−159/−125; Phospholamban Promoter Element 1, PPE1) required for promoter activity in cardiomyocytes. Site-specific mutations identified nucleotides −150/−133 as containing most of the promoter-enhancing activity. While the rat PPE1 is highly conserved (>70%) in four other mammalian phospholamban genes, it does not contain previously characterized regulatory elements. In cardiomyocytes the PPE1 sequence markedly enhanced activity of the SV40 early promoter. A conserved CCAAT element (−83/−79) was also required for promoter activity in both cardiomyocytes and HeLa cells. Exonuclease III footprinting identified protein/DNA interactions in both the extended CCAAT box and PPE1 domains. Gel shift studies identified the CCAAT elements as binding CBF/NF-Y.

The Tarsius γ-Globin Gene: Pseudogene or Active Gene?

We sequenced the ∼5-kb long γ-globin gene locus from Tarsius bancanus and compared it to the published γ-globin gene sequence from the related species Tarsius syrichta. The T. syrichta gene's promoter lacks the distal CCAAT box and has a point mutation in the functionally important proximal CCAAT box (CCgAT). This previous finding had suggested that in tarsiers the γ-globin gene might be a nonexpressed pseudogene. The two tarsier species show the same point mutation at the third nucleotide of the proximal CCAAT element and absence of the distal CCAAT element. Nevertheless, our results indicate that in tarsiers the γ-globin gene is active, since all three coding regions show only synonymous substitutions and a much lower level of divergence than the noncoding regions. This is significantly different from what would be expected for a silent gene evading stabilizing selection. Thus, we hypothesize that the tarsier's γ-globin gene locus is expressed even with the mutation in the proximal CCAAT box.

Analysis of DNA elements that modulate myosin VIIA expression in humans.

Usher syndromeIb (USH1B), an autosomal recessive disorder caused by mutations in myosin VIIa (MYO7A), is characterized by congenital profound hearing loss, vestibular abnormalities and retinitis pigmentosa. Promoter elements in the 5 kb upstream of the translation start were identified using adult retinal pigment epithelium cells (ARPE-19) as a model system. A 160 bp minimal promoter within the first intron was active in ARPE-19 cells, but not in HeLa cells that do not express MYO7A. A 100 bp sequence, 5' of the first exon, and repeated with 90% homology within the first intron, appeared to modulate expression in both cell lines. Segments containing these elements were screened by heteroduplex analysis. No heteroduplexes were detected in the minimal promoter, suggesting that this sequence is conserved. A -2568 A>T transversion in the 5' 100 bp repeat, eliminating a CCAAT element, was found only in USH1B patients. However, in all 5 families, -2568 A>T was in cis with the same missense mutation in the myosin VIIa tail (Arg1240Gln), and 4 of the 5 families were Dutch. These observations suggest either 1) linkage disequilibrium or 2)that a combination of a promoter mutation with a less active myosin VIIa protein results in USH1B.

Interaction between the −170 CRE and the −150 CCAAT box is necessary for efficient activation of the fibronectin gene promoter by cAMP and ATF-2

The fibronectin promoter contains an ATF/cyclic AMP (cAMP) response element (CRE) site two helical turns upstream of a CCAAT site with which it interacts. We investigated the effects of mutating these −170 CRE and −150 CCAAT elements on the promoter activity regulated by three different modulators previously known to act through CRE: ATF-2, cAMP and E1a. While the cooperation seems to play no role in E1a action, integrity of the −150 CCAAT is necessary for ATF-2 and cAMP efficient activation in a cell-specific manner. These results show that the CRE and CCAAT elements function as a ‘composite element’ and establish a cell-specific function for CRE-CCAAT synergy.

Transcriptional activation of E2F1 gene expression by 17beta-estradiol in MCF-7 cells is regulated by NF-Y-Sp1/estrogen receptor interactions.

17beta-Estradiol (E2) stimulated proliferation and DNA synthesis in MCF-7 human breast cancer cells, and this was accompanied by induction of E2F1 mRNA and protein levels. Analysis of the E2F1 gene promoter showed that the -146 to -54 region was required for E2-responsiveness in transient transfection assays, and subsequent deletion/mutation analysis showed that a single upstream GC-rich and two downstream CCAAT-binding sites were required for transactivation by E2. Gel mobility shift assays with multiple oligonucleotides and protein antibodies (for supershifts) showed that the -146 to -54 region of the E2F1 gene promoter bound Sp1 and NF-Y proteins in MCF-7 cells. The estrogen receptor (ER) protein enhanced Sp1 interactions with upstream GC-rich sites, and interactions of ER, Sp1, and ER/Sp1 with downstream DNA bound-NF-Y was investigated by kinetic analysis for protein-DNA binding (on- and off-rates), coimmunoprecipitation, and pulldown assays using wild-type and truncated glutathione S-transferase (GST)-Sp1 chimeric proteins. The results showed that Sp1 protein enhanced the Bmax of NF-Y-DNA binding by more than 5-fold (on-rate); in addition, the Sp1-enhanced NF-Y-DNA complex was further stabilized by coincubation with ER and the rate of dissociation (t1/2) was decreased by approximately 50%. Sp1 antibodies immunoprecipitated [35S]NF-YA after coincubation with unlabeled Sp1 protein. Thus, transcriptional activation of E2F1 gene expression in MCF-7 cells by E2 is regulated by multiprotein ER/Sp1-NF-Y interactions at GC-rich and two CCAAT elements in the proximal region of the E2F1 gene promoter. This represents a unique trans-acting protein complex in which ligand-dependent transactivation by the ER is independent of direct ER interactions with promoter elements.

Cell Cycle-dependent Switch of Up- and Down-regulation of Human hsp70 Gene Expression by Interaction between c-Myc and CBF/NF-Y

A CCAAT box-binding protein subunit, CBF-C/NF-YC, was cloned as a protein involved in the c-Myc complex formed on the G(1)-specific enhancer in the human hsp70 gene. CBF-C/NF-YC directly bound to c-Myc in vitro and in vivo in cultured cells. The CBF/NF-Y.c-Myc complex required the HSP-MYC-B element as well as CCAAT in the hsp70 G(1)-enhancer, while the purified CBF subunits recognized only CCAAT even in the presence of c-Myc. Both the HSP-MYC-B and CCAAT elements were also required for the enhancer activity. In transient transfection experiments, the CBF/NF-Y.c-Myc complex, as well as transcription due to the G(1)-enhancer, was increased by the introduction of c-Myc at low doses but decreased at high doses. The repression of both complex formation and transcription by c-Myc at high doses was abrogated by the introduction of CBF/NF-Y in a dose-dependent manner. Furthermore, the CBF/NF-Y.c-Myc complex bound to the G(1)-enhancer appeared in the early G(1) phase of the cell cycle when c-Myc was not higly expressed and gradually disappeared after the c-Myc expression reached its maximum. The results indicate that the cell cycle-dependent expression of the hsp70 gene is regulated by the intracellular amount of c-Myc through the complex formation states between CBF/NF-Y and c-Myc.

Hormonal regulation and genomic organization of the human amiloride-sensitive epithelial sodium channel alpha subunit gene.

To investigate the regulation of the amiloride-sensitive epithelial sodium channel (ENaC) expression, we have characterized the genomic structure and performed promoter analyses of the alpha subunit of the human (h) ENaC gene. Genomic clones containing the alphahENaC gene were isolated and subjected to restriction-mapping analysis. The alphahENaC gene was shown to be composed of 13 exons and 12 introns. Primer extension analysis confirmed that transcription initiation occurred at the beginning of the reported alphahENaC cDNA, but also indicated potential heterogenous initiation sites. Examination of a 3.1 kb 5' flanking sequence revealed a notable absence of CCAAT or TATA-like elements but suggested three GC boxes and several putative transcription factor binding sites, including a glucocorticoid response element (GRE) consensus. A 250 bp minimal promoter was capable of directing expression of a secreted alkaline phosphatase reporter. This promoter activity was enhanced 2.5- and 4-fold by upstream flanking sequences. Dexamethasone treatment induced levels of expression from the longer, GRE-containing promoter fragments from 8- to 20-fold, but not from the minimal promoter. Precise deletion of the 15-bp, dyad GRE sequence completely abolishes the response of reporter expression to dexamethasone induction. These experiments indicate that glucocorticoid augmentation of lung epithelial Na+ transport occurs, at least in part, by direct stimulation of transcription of the ENaC genes.

TATA-driven transcriptional initiation and regulation of the rat serotonin 5-HT1A receptor gene.

The transcriptional initiation and regulation of the rat serotonin 5-HT1A receptor gene were characterized. By three types of analyses, a single brain-specific site of transcriptional initiation was localized to -967 bp upstream of the translation initiation codon that is utilized both in hippocampus and in the rat raphe RN46A cell line. This major site of transcriptional initiation was located 58 bp downstream from a consensus TATA element, suggesting TATA-driven transcription of the rat 5-HT1A receptor. To identify the promoter activity of the receptor gene, progressive 5' deletions of the -2,719/-117-bp fragment of the 5-HT1A promoter linked to luciferase gene were transfected into 5-HT1A-negative (pituitary GH4C1, L6 myoblast, and C6 glioma) and 5-HT1A-positive (septal SN-48 and raphe RN46A) cell lines. Enhancer regions were identified within a fragment between nucleotides -426 and -117 that selectively enhanced transcription in 5-HT1A-positive cells. A nonselective enhancer/promoter that mediated expression in all cell lines was located upstream between -1,519 and -426 bp in a DNA segment containing consensus TATA, CCAAT, SP-1, and AP-1 elements as well as a poly-GT26 dinucleotide repeat. Strong repression of transcription in all cell lines was conferred by the region upstream of -1,519 bp that contains a 152-bp DNA segment with >80% identity to RANTES, tumor necrosis factor-beta, and other immune system genes. Our results indicate that TATA-driven expression of the 5-HT1A receptor is regulated by a novel proximal tissue-specific enhancer region, a nonselective promoter, and an upstream repressor region that is distinct from previously identified neuron-specific repressors.