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Perspectives on the RNA polymerase II core promoter.

The RNA polymerase II core promoter is a critical yet often overlooked component in the transcription process. The core promoter is defined as the stretch of DNA, which encompasses the RNA start site and is typically approx. 40-50 nt in length, that directs the initiation of gene transcription. In the past, it has been generally presumed that core promoters are general in function and that transcription initiation occurs via a common shared mechanism. Recent studies have revealed, however, that there is considerable diversity in core promoter structure and function. There are a number of DNA elements that contribute to core promoter activity, and the specific properties of a given core promoter are dictated by the presence or absence of these core promoter motifs. The known core promoter elements include the TATA box, Inr (initiator), BRE(u) {BRE [TFIIB (transcription factor for RNA polymerase IIB) recognition element] upstream of the TATA box} and BRE(d) (BRE downstream of the TATA box), MTE (motif ten element), DCE (downstream core element) and DPE (downstream core promoter element). In this paper, we will provide some perspectives on current and future issues that pertain to the RNA polymerase II core promoter.

Models, Genetic↗

Control elements between -9.5 and -3.0 kb in the human tissue-type plasminogen activator gene promoter direct spatial and inducible expression to the murine brain.

Tissue-type plasminogen activator (t-PA) participates in the control of synaptic plasticity and memory formation in the central nervous system (CNS). Transgenic mice harbouring either 9.5, 3.0 or 1.4 kb of the human t-PA promoter fused to the LacZ reporter gene were used to assess t-PA promoter-directed expression in vivo. The 9.5 kb t-PA promoter directed expression to the brain, most notably to the dentate gyrus, superior colliculus, hippocampus, thalamus and piriform cortex. Staining was also observed in the retrosplenial and somatosensory cortex. The 3.0 kb t-PA promoter directed generalized and poorly defined expression to the cortex and hippocampus, while the 1.4 kb t-PA promoter directed expression selectively to the medial habenula. Intravenous administration of lipopolysaccharide into mice harbouring the 9.5 kb t-PA promoter resulted in an increase in reporter gene activity in the lateral orbital cortex and thalamus. Results of in vitro transfection experiments of NT2 cells with a series of t-PA promoter deletion constructs confirmed the presence of regulatory elements throughout the 9.5 kb promoter region. Finally, we describe a cis-acting element related to the NFAT recognition site that provides a protein-binding site and which may play a role in the selective expression of the 1.4 t-PA promoter in the medial habenula. These results indicate that elements between -3.0 and -9.5 kb of the t-PA promoter confer constitutive and inducible expression to specific regions of the CNS.

Animals↗

Roles of multiple promoter elements of the proliferating cell nuclear antigen gene during Drosophila development.

The expression of genes involved in DNA replication is closely correlated with the proliferating state of cells and is repressed with the progression of differentiation during development. Promoter regions of the Drosophila proliferating cell nuclear antigen (PCNA) gene and the DNA polymerase alpha gene contain a common 8-base pair promoter element (DRE: DNA replication-related element). The examination of a common expression mechanism for DNA replication-related genes, which is regulated positively by growth signals and negatively by differentiation signals would be of interest. We generated PCNA-LacZ fusion genes in which the 5'-flanking sequence of the PCNA gene has been mutated. An examination of the expression of these fusion genes, introduced into flies by germ-line transformation, led to the identification of another distinct regulatory element, URE (upstream regulatory element), within the region from -168 to -119 with respect to the transcription initiation site. During embryogenesis, the region containing the DRE sequence (-108 to -91) greatly stimulated the PCNA gene minimal promoter (-86 to +130), when it was placed upstream of the promoter in both normal and reverse orientations. Addition of the URE sequence further stimulated the promoter activity twofold. During larval stages, both DRE and URE were indispensable to the promoter activity, since neither of the sequences alone activated the minimal promoter. Demonstration of beta-galactosidase activity indicated URE plays an essential role in various larval tissues such as salivary gland and imaginal disc. While the minimal promoter region alone directed maternal expression of lacZ in ovaries of adult females, both DRE and URE further stimulated promoter activity. These results show several elements of the PCNA gene promoter play roles during Drosophila development.

Animals↗

Health promotion training needs analysis: an integral role for clinical nurses in Lanarkshire, Scotland.

Training needs analysis is considered essential to professional and organizational development, for continuing education and for incorporation into professional practice. Increasingly, nurses are encouraged to develop their health promotion role. Despite this there has been relatively little research in Scotland into the health promotion role of the hospital nurse. This study describes the results of a health promotion training needs analysis undertaken on qualified, hospital-based nurses in Lanarkshire, Scotland. Information on the nurses' current health promotion practices, their attitudes and beliefs, their views on role development and priorities for further training were collated, analysed and discussed within the framework of the Health Action Model. This model was originally designed to assist understanding of the gap that exists between an individual intention to act and the eventual health action. However, in this study the ways in which 'cognitive factors' (knowledge and beliefs) and 'motivational factors' (considering values, attitudes and drives) and pressures from social norms and significant others assisted in the understanding of the nurse's orientation to, and beliefs about, health promotion practice. Fifty-seven per cent of nurses in the study considered health promotion activities to be emerging in clinical care and 4% considered them to be advanced. The interest respondents have in role development and the further integration of health promotion activities into nursing practice was explored by ascertaining the nurses' attitudes and beliefs about their own health promotion role. Of 107 respondents, a majority 72 (67%) agreed health promotion interventions to be an important function of the nurse with 29 (27%) strongly agreeing (n = 107). Sixty per cent stated they would be interested in developing their role and 30% were very strongly interested. Recommendations are therefore made for future planning and development strategies for the health promotion role of hospital nurses.

Adult↗

Health promotion projects: skill and attitude learning for medical students.

OBJECTIVES: The aim of this health promotion project is to introduce students to appropriate skills and attitudes--as well as knowledge about health promotion strategies and methods. As part of this process, standardized procedures have been established to ensure that the projects are scientifically and ethically appropriate and adequately supervised. This project-centred course introduces the discipline of health promotion to third-year medical students at Monash University. It is aimed at introducing students to the range of health promotion concepts, providing them with experience of health promotion activities and involving them in consideration of the scientific, political and ethical issues arising from doctors' participation in health promotion. DESIGN: As the major learning and assessment component of the unit, students participate in self-selected project groups of three to five students. Each group develops a topic for a health promotion activity in the community, carries out that project and presents the results as a poster as well as a written report. SETTING: Monash University. SUBJECTS: Third-year medical students. RESULTS: Sixty per cent of each student's mark for the unit is based on the project. The posters produced by the project groups are placed on public display in a major teaching hospital for a week at the end of the unit. Public display of the posters helps each student to appreciate the variety of possible health promotion activities, and to appreciate health promotion as a scientific discipline. It also makes the project findings available to the public. CONCLUSIONS: Student evaluation of the project, and community response to the projects--especially the poster display-- indicate that the project is both a highly effective learning experience and a health-promoting activity in its own right.

Attitude↗

Molecular characterization of the pH-inducible and growth phase-dependent promoter P170 of Lactococcus lactis.

In a previous study, we described the use of transposon Tn917-LTV1 for identification of environmentally regulated promoters in Lactococcus lactis. Here, we report the molecular analysis of one of these promoters, P170, that is upregulated at low pH during the transition to stationary phase. The minimal DNA region required for both promoter activity and pH regulation was mapped to a 51 bp fragment located 7 bp upstream of the transcriptional start site. This fragment lacked the consensus -35 promoter region, but it contained an 'extended' -10 promoter region. When a 28 bp segment, containing the consensus -35 region and 22 bp upstream of this in a constitutive promoter, was replaced with the corresponding sequence of P170, the hybrid promoter became regulated by pH and growth phase. This demonstrates that the P170 segment contains a cis-acting sequence involved in the control of promoter regulation. Transcriptional analysis showed that P170 is responsible for the transcription of a monocistronic gene orfX encoding a polypeptide homologous to a hypothetical protein from Bacillus subtilis. Analysis of total RNA from L. lactis grown at constant pH confirmed that transcription from P170 was induced between pH 6.5 and pH 6.0, but only when the culture entered stationary phase. Deletion analysis and chemical mutagenesis of P170 defined a specific region within the untranslated mRNA leader that is able to modulate the expression level directed by the P170 promoter. Deletion of a 72 bp HaeIII fragment from this leader region resulted in a 150- to 200-fold increase in the level of gene expression, without affecting the regulation. The functionality was confirmed by introducing this modulating element downstream of other lactococcal promoters.

Base Sequence↗

ResD signal transduction regulator of aerobic respiration in Bacillus subtilis: ctaA promoter regulation.

A two-component signal transduction system composed of a sensor kinase, ResE, and a response regulator, ResD, encoded by resD and resE genes of the res operon (resABCDE), has a regulatory role in both aerobic and anaerobic respiration. In terms of aerobic respiration, resD functions upstream of ctaA, a gene required for haem A biogenesis and hence for the synthesis of haem A-containing cytochrome terminal oxidases. Although ResD is probably a transcription factor, there was no direct evidence that ResD protein, either phosphorylated or unphosphorylated, interacts directly with regulatory regions of ResD-controlled genes. Here, we report the overexpression and purification of ResD and ResE and their role in gene activation. ResD can be phosphorylated by ResE in vitro and is a monomer in solution in either the phosphorylated or unphosphorylated state. The binding activity of ResD to the ctaA promoter was examined by gel shift assays and DNase I footprinting assays. DNase I footprinting showed both unphosphorylated and phosphorylated ResD binding to the ctaA promoter and showed that there are three binding sites (A1, A2 and A3), two (A1 and A2) upstream of the -35 promoter region and one (A3) downstream of the -10 of the promoter. The role of each site in ctaA promoter activity and ResD binding was characterized using deletion analysis, followed by the DNase I footprinting and in vivo transcription assays of promoter-lacZ fusions. Our results showed that the concentration of ResD required to bind at each site is different and that ResD binding at the A1 site is independent of the other two ResD binding sites, but that the concentration of ResD approximately P required to protect site A2 is reduced when site A3 is present. In vivo transcription assays from promoter-lacZ fusion constructs showed that DNA containing ResD-binding site A2 was essential for promoter activity and that promoter constructs containing both binding sites A2 and A3 were sufficient for full promoter activity.

Aerobiosis↗

Expression of the rabbit cytochrome P450 aromatase encoding gene uses alternative tissue-specific promoters.

The aim of the present study was to analyse the tissue-specific expression of various promoter-derived transcripts from the gene encoding rabbit aromatase cytochrome P450. A new promoter, named I.r, was identified, and promoters II and I.r were sequenced. Promoter I.r-derived transcripts were found in preovulatory granulosa cells, corpus luteum, placenta and adipose tissue. An alternative splice variant of this transcript was found with tissue-specific preference. Tissue-specific expression of promoter-derived variants was studied in the ovary before and after ovulation. While the level of promoter II-derived transcript decreased dramatically after ovulation, that of promoter I.r-derived transcript remained unchanged, indicating that promoter II and promoter I.r were not controlled by a single regulation system. The existence of this dual system of regulation suggests that the rabbit ovary could be a useful model to study the promoter-specific regulation of aromatase.

Alternative Splicing↗

Transcriptional regulation of human excitatory amino acid transporter 1 (EAAT1): cloning of the EAAT1 promoter and characterization of its basal and inducible activity in human astrocytes.

Excitatory amino acid transporter 1 (EAAT1) is one of the two glial glutamate transporters that clear the extracellular glutamate generated during neuronal signal transmission. Here, we cloned and characterized a 2.1-kb promoter region of human EAAT1 and investigated its function in the transcriptional regulation of the EAAT1 gene in human primary astrocytes. The full-length promoter region lacked TATA and CCAAT boxes and an initiator element, it contained several potential transcription factor-binding sites and it exhibited promoter activity in primary astrocytes and in several types of transformed cells. Consecutive 5'-deletion analysis of the EAAT1 promoter indicated the presence of negative and positive regulatory regions and a putative core promoter between -57 bp and +20 bp relative to the transcription start site (TSS). The core promoter contained a single GC-box in position -52/-39 and one E-box near the TSS and the GC-box site that was responsible for 90% of the basal promoter activity as determined by mutational analysis. Electrophoretic mobility shift, supershift and competition assays demonstrated binding of stimulating proteins (Sp) 1 and 3 to the GC-box and upstream stimulating factor (USF) 1 to the E-box. Treatment of primary human astrocytes with cellular modulators 8-bromo cyclic AMP and epidermal growth factor increased EAAT1 promoter activity in transient transfection assays and increased cellular EAAT1 mRNA expression and glutamate uptake by astrocytes. Conversely, tumor necrosis factor-alpha reduced both EAAT promoter activity and cellular EAAT1 mRNA expression. These results enable studies of transcriptional regulation of EAAT1 gene at the promoter level.

8-Bromo Cyclic Adenosine Monophosphate↗

Regulation of human renin gene promoter activity: a new negative regulatory region determines the responsiveness to TNF alpha.

BACKGROUND: The renin-angiotensin system has been known to regulate blood pressure and body fluid homeostasis. Several lines of evidence have shown that renin gene expression and release are up-regulated by beta-adrenergic stimulation, sodium depletion, and angiotensin converting enzyme inhibition, but down-regulated by cytokines. To further characterize the human renin gene (hREN) promoter structure, its regulation, and to identify an appropriate cell system for study, we examined five cell lines and investigated drug effects on the hREN promoter expression. METHODS: Using the hREN-luciferase reporter gene constructs in the DNA transfection assays, approximately 5 kb of the hREN 5' flanking region was assessed for promoter activity in five different cell lines. Regulation of the hREN promoter activity was investigated using Y-1 adrenal cells that were transfected with the hREN-luciferase DNA and were treated with forskolin, calcium ionophore A23187, phorbol ester, angiotensin II (Ang II), or cytokines. RESULTS: Transient transfection analysis showed that the 5 kb hREN 5' flanking DNA alone was able to confer significant promoter activity in Y-1 adrenal cells. In transfected Y-1 cells, luciferase reporter expression was induced by forskolin, suppressed by the calcium ionophore A23187, and phorbol ester in a dose-dependent manner, but was unaffected by angiotensin II (Ang II). However, when Y-1 reporter cells were transfected with human angiotensin II receptor type 1 (AT1) cDNA, hREN promoter activity was dose-dependently down-regulated by Ang II, which was blockable by losartan, an AT1-selective antagonist. Further studies also showed that hREN promoter activity in Y-1 cells was selectively down-regulated by TNF alpha. Deletion of the hREN promoter sequences between position -3916 and -2822 not only enhanced hREN promoter activity by approximately tenfold, but also caused a failure of down-regulation by TNF alpha. In contrast, neither interleukin (IL)-1 alpha, IL-1 beta, IL-2, nor IL-6 exerted any significant effect. CONCLUSIONS: Together the results suggest that TNF alpha is a negative regulator of the hREN expression in the adrenal cells, and that the TNF alpha responsiveness may be controlled by elements located between the positions -3916 and -2822 of the hREN promoter. Moreover, the Y-1 cell line may provide a valuable model system for studying renin gene regulation.

Adrenal Glands↗

Transient expression in mammalian cells of transgenes transcribed from the Cauliflower mosaic virus 35S promoter.

Gene constructs containing the Cauliflower mosaic virus (CaMV) 35S promoter and a sequence coding either for a green fluorescent protein (GFP) or for firefly luciferase were transfected into Chinese hamster ovary (CHO) cells. Both reporter genes were expressed to significant levels. The 35S promoter was 40 times less active than the human eF1 alpha promoter, which is known to be one of the most potent promoters in mammalian cells. The 35S promoter must therefore be considered to be a promoter of significant potency in mammalian cells. RT-PCR analysis suggested that transcription initiation in CHO cells occurred between the TATA box and the transcription start site of the 35S promoter that function in plant cells. Further analysis by 5'RACE confirmed that transcription was initiated in CHO cells at different sites located essentially between the TATA box and the plant transcription start site, showing that 35S promoter activity in animal cells is due to the presence of promoter elements that are functional in mammalian cells, but that are not those used in plants. The data reported here raise the possibility that genes controlled by the 35S promoter, which is commonly used in transgenic plants, have the potential for expression in animal cells.

Animals↗

Importance of Sp1 consensus motifs in the MYCN promoter.

BACKGROUND: MYCN (N-myc) amplification in neuroblastoma is associated with poor clinical outcome. Factors that regulate MYCN expression have not been elucidated. MYCN is considered a TATA-less promoter, whereas significant promoter activity resides within 160 bp 5' of the major transcription start site. This region contains two GC-rich motifs and a CT box, regions for potential transcription factor interaction. METHODS: To characterize DNA-protein interactions in this region of the MYCN promoter, electrophoretic mobility shift assays, and promoter-reporter were used. RESULTS: A MYCN promoter fragment was incubated with HeLa nuclear extract, with or without competitors. Three major protein/DNA complexes were formed. Formation of 2 complexes could be inhibited by unlabeled Sp1 consensus duplex and by the Sp1 site-specific drug WP631. Purified Sp1 protein produced a complex similar to that formed with HeLa extract. To determine whether these DNA/protein interactions could be blocked in a sequence-specific fashion, a triplex forming oligonucleotide (TFO) was used. This TFO was designed to bind in the major groove of the promoter, covering the CT-box (putative Sp1 binding) motif. When triplex formation was followed by addition of nuclear extract, protein binding was indeed inhibited. Functional significance of this inhibition was tested with pE/Bnmyc-luc, a promoter-reporter plasmid containing the human MYCN promoter driving luciferase expression. Incubation with TFO, but not control oligodeoxynucleotides, completely inhibited luciferase activity. CONCLUSIONS: These data suggest that protein binding does occur in regions of the MYCN promoter containing GC and CT box elements and that this interaction is important for MYCN promoter activity. By inference, these data also suggest that the proteins that bind in this region are Sp1 family members.

Base Sequence↗

Sequence-independent upstream DNA-alphaCTD interactions strongly stimulate Escherichia coli RNA polymerase-lacUV5 promoter association.

The C-terminal domains of the two alpha-subunits (alphaCTD) in Escherichia coli RNA polymerase (RNAP) recognize specific sequences called UP elements in some promoters. These interactions can increase transcription dramatically. Previously, effects of upstream DNA-alphaCTD interactions on transcription were quantified relative to control promoters with nonspecific DNA sequences substituted for UP elements. However, contributions of nonspecific upstream DNA-alphaCTD interactions to promoter activity have not been evaluated extensively. Here, we examine effects of removal of alphaCTD, upstream promoter DNA, or both on the rate of open-complex formation with promoters that lack UP elements. Deletion of alphaCTD decreased the composite second-order association rate constant, k(a), of RNAP for the lacUV5 promoter by approximately 10-fold. Much of this effect was attributable to a decrease in the isomerization rate constant, k(2). Removal of promoter DNA upstream of the -35 element also decreased both k(a) and k(2) approximately 10-fold. Upstream DNA extending approximately to base pair -100 was sufficient for maximal association rates of wild-type RNAP with lacUV5 promoter fragments. The alphaCTD and upstream DNA did not affect dissociation rates from the open complex. We suggest that sequence-independent upstream DNA interactions with alphaCTD are major contributors to initiation at many (or all) promoters (not merely promoters containing UP elements) and that these interactions facilitate isomerization events occurring well downstream of the alpha-binding sites. In addition to highlighting the functional importance of nonspecific protein-DNA interactions, these results suggest also that UP element-alphaCTD interactions play an even larger role in transcription initiation than appreciated previously.

Base Sequence↗

Genomic sequencing reveals a 5-methylcytosine-free domain in active promoters and the spreading of preimposed methylation patterns.

Previous work demonstrated an inverse correlation between methylation at the three 5'-CCGG-3' sequences in positions +24, +6, and -215 relative to the cap site of the late E2A promoter of adenovirus type 2 (Ad2) DNA and its activity. In the study presented here, we used the genomic sequencing method to detect 5-methyl-2'-deoxycytidine (m5dC) residues in 5'-CG-3' sequences other than the 5'-CCGG-3' (Hpa II) sites. The patterns of methylation in all 5'-CG-3' sequences over a region of about 180 base pairs required for gene activity in the late E2A promoter of integrated Ad2 DNA were determined in cell lines that carry this promoter in an active or inactive state. In cell lines HE1 and uc2, the late E2A promoter is active and all thirteen 5'-CG-3' sequences between positions +24 and -160 are unmethylated. In cell line HE2, the same promoter is permanently shut off and all 5'-CG-3' sequences are methylated in both strands. Thus, the inverse correlation is perfect in these cell lines over a region of about 180 base pairs in the late E2A promoter. The same promoter segment was analyzed in cell lines mc23 and mc40, in which a late E2A promoter-chloramphenicol acetyltransferase (CAT) gene construct had been genomically fixed after in vitro 5'-CCGG-3' methylation and subsequent transfection. In cell line mc23, the preimposed methylation pattern was stable and the CAT gene was inactive. Genomic sequencing confirmed the presence of m5dC in the 5'-CCGG-3' sequences and revealed the spreading of methylation to neighboring 5'-CG-3' sequences along the entire promoter. Some of these sites were hemimethylated. In cell line mc40, several of the 120 integrated copies became demethylated in positions +24 and +6, but the promoter was methylated in some of the copies upstream of position -50. Cell line mc40 expressed the CAT gene.

5-Methylcytosine↗

Functional binding of the "TATA" box binding component of transcription factor TFIID to the -30 region of TATA-less promoters.

Many viral and cellular promoters transcribed in higher eukaryotes by RNA polymerase II lack obvious A+T-rich sequences, called "TATA" boxes, that bind the transcription factor TFIID. One such TATA-less promoter, the simian virus 40 major late promoter, contains a genetically important sequence element 30 base pairs upstream of its transcription initiation site that has no obvious sequence similarity to a TATA box. We show here that the cloned human TATA box-binding protein, hTFIID tau, functionally binds to this upstream sequence element, although with an affinity one-sixth of that to which it binds the TATA box of the adenovirus type 2 major late promoter. Analysis of point mutations in the -30 element of the simian virus 40 major late promoter shows that the affinity of binding correlates with the efficiency of transcription from this promoter. Furthermore, this element has genetic properties similar to those of a TATA box. (i) It directs RNA polymerase II to initiate transcription approximately 30 base pairs downstream of its location, and (ii) inactivation of this element results in increased heterogeneity in the sites of transcription initiation. All of five other TATA-less promoters tested were found to contain a sequence approximately 30 base pairs upstream of their major transcription initiation sites to which hTFIID tau binds. We conclude that many, if not all, TATA-less promoters differ from TATA box-containing promoters simply in the affinity of their -30 regions for binding of TFIID, with functional binding of TFIID supported in part by other nearby sequence elements of the promoter.

Base Sequence↗

Activation of the cytotactin promoter by the homeobox-containing gene Evx-1.

Cytotactin is a morphoregulatory molecule of the extracellular matrix affecting cell shape, division, and migration that appears in a characteristic and complex site-restricted pattern during embryogenesis. The promoter region of the gene that encodes chicken cytotactin contains a variety of potential regulatory sequences. These include putative binding sites for homeodomain proteins and a phorbol 12-O-tetradecanoate 13-acetate response element (TRE)/AP-1 element, a potential target for transcription factors thought to be involved in growth-factor signal transduction. To determine the effects of homeobox-containing genes on cytotactin promoter activity, we conducted a series of cotransfection experiments on NIH 3T3 cells using cytotactin promoter-chloramphenicol acetyltransferase (CAT) reporter gene constructs and plasmids driving the expression of mouse homeobox genes Evx-1 and Hox-1.3. cotransfection with Evx-1 stimulated cytotactin promoter activity whereas cotransfection in control experiments with Hox-1.3 had no effect. To localize the sequences required for Evx-1 activation, we tested a series of deletions in the cytotactin promoter. An 89-base-pair region containing a consensus TRE/AP-1 element was found to be required for activation. An oligonucleotide segment containing this TRE/AP-1 site was found to confer Evx-1 inducibility on a simian virus 40 minimal promoter; mutation of the TRE/AP-1 site abolished this activity. To explore the potential role of growth factors in cytotactin promoter activation, chicken embryo fibroblasts, which are known to synthesize cytotactin, were first transfected with cytotactin promoter constructs and cultured under minimal conditions in 1% fetal bovine serum. Although the cells exhibited only low levels of CAT activity under these conditions, cells exposed for 12 h to 10% (vol/vol) fetal bovine serum showed a marked increase in CAT activity. Cotransfection with Evx-1 and cytotactin promoter constructs of cells cultured in 1% fetal bovine serum was sufficient, however, to produce high levels of CAT activity. These findings are consistent with the hypothesis that Evx-1, a homeobox-containing gene, may activate the cytotactin promoter by a mechanism involving a growth-factor signal transduction pathway. More generally, the results support the hypothesis that the place-dependent expression of morphoregulatory molecules may depend upon local cues provided by homeobox genes and their encoded proteins.

3T3 Cells↗

Maize Spm transposable element has an enhancer-insensitive promoter.

We have used a transient assay system to investigate the promoter region of the maize Suppressor-mutator (Spm) transposable element. All of the sequence required for constitutive promoter activity is confined to the 0.2-kb sequence upstream from the transcription start site of the element at nt 209 and designated the upstream control region. The element's promoter is weak, lacks a conventional TATA box, and depends on the presence of multiple, short repetitive sequence elements. The Spm promoter is quite insensitive to the enhancer sequence of the cauliflower mosaic virus 35S promoter. Enhancer sensitivity can be restored by providing a -30 TATA sequence and removing the G + C-rich sequence encoding the untranslated leader of the element, designated the downstream control region. Although the downstream control region is without effect on Spm promoter activity, it completely inhibits the 35S core promoter and markedly inhibits activity of the complete 35S promoter. The properties of the Spm element's promoter buffer it from both mutational and position-dependent changes in activity. We suggest that the inherent characteristics of the promoter are part of the genetic mechanism that controls the element's transposition frequency, ensuring it remains low and insertion-site independent.

Base Sequence↗

Binding and transcriptional activation of the promoter for the neural cell adhesion molecule by HoxC6 (Hox-3.3).

Scores of homeobox gene-encoded transcription factors are expressed in a definite spatiotemporal pattern during embryogenesis and regulate a series of as yet unidentified target genes to help coordinate the morphogenetic process. We have suggested that homeobox gene products modulate the expression of adhesion molecule genes and have shown in cotransfection experiments that the promoters for the neural cell adhesion molecule (N-CAM) and cytotactin/tenascin genes respond to cues from different homeobox-containing genes. In this study, we show that the HoxC6 (Hox-3.3)-encoded homeoprotein binds to a DNA sequence in the N-CAM promoter CCTAATTATTAA, designated homeodomain binding site I (HBS-I). To test whether HoxC6 regulated N-CAM promoter activity, we cotransfected the Long and Short reading frame variants of Xenopus HoxC6 (CMV-HoxC6-L and CMV-HoxC6-S) driven by the human cytomegalovirus (CMV) promoter together with a chloramphenicol acetyltransferase (CAT) reporter gene driven by the mouse N-CAM promoter (N-CAM-Pro-CAT). Cotransfection of NIH 3T3 cells with either of the CMV-HoxC6 expression vectors stimulated N-CAM promoter-driven CAT expression. A 47-bp region from the N-CAM promoter that included HBS-I and an adjacent potential HBS, HBS-II, conferred HoxC6 regulation on a simian virus 40 minimal promoter. HBS-I was sufficient for transactivation of the minimal promoter by CMV-HoxC6-S. However, transcriptional activation by CMV-HoxC6-L required both HBS-I and HBS-II, inasmuch as mutation of either HBS-I, HBS-II, or both motifs abolished the response. These studies suggest that HBS-I is a target site for binding and transcriptional control of the N-CAM promoter by homeoproteins, although accessory DNA sequences (such as HBS-II) may also be required. Together with previous studies, these results support the notion that N-CAM gene expression may be controlled by different combinations of homeoproteins that appear in a place-dependent manner during embryogenesis.

3T3 Cells↗

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