SV40 Enhancer Research Articles

Human Chorionic Somatomammotropin Enhancer Function Is Mediated by Cooperative Binding of TEF-1 and CSEF-1 to Multiple, Low-Affinity Binding Sites

The human chorionic somatomammotropin gene enhancer (CSEn) is composed of multiple enhansons (Enh) that share sequence similarities with those of the simian virus, SV40 enhancer (SVEn). The sequence homology includes two GT-IIC-like (Enh1 and Enh4) and three SphI/II-like enhansons (Enh2, Enh3, and Enh5). We previously showed that transcription enhancer factor 1 (TEF-1) and a 30-kDa placental-specific factor, chorionic somatomammotropin enhancer factor 1 (CSEF-1), bind to Enh4, which plays an essential role in enhancer function. In this study, we demonstrate that TEF-1 and CSEF-1 bind specifically to all the other GT-IIC- and SphI/II-like elements within CSEn with a broad range of binding affinities that vary between 0.005 and 0.15 that of Enh4. Each individual concatenated enhanson was able to stimulate hCS promoter activity in an orientation-independent manner in choriocarcinoma cells (BeWo) with an observed stimulation that was directly proportional to its relative binding affinity for TEF-1 and CSEF-1. These results indicate that CSEn function results from the cooperative interaction of TEF-1 and/or CSEF-1 binding to multiple, low-affinity GT-IIC- and SphI/II-like enhansons within the enhancer.

Human chorionic somatomammotropin enhancer function is mediated by cooperative binding of TEF-1 and CSEF-1 to multiple, low-affinity binding sites.

The human chorionic somatomammotropin gene enhancer (CSEn) is composed of multiple enhansons (Enh) that share sequence similarities with those of the simian virus, SV40 enhancer (SVEn). The sequence homology includes two GT-IIC-like (Enh1 and Enh4) and three SphI/II-like enhansons (Enh2, Enh3, and Enh5). We previously showed that transcription enhancer factor 1 (TEF-1) and a 30-kDa placental-specific factor, chorionic somatomammotropin enhancer factor 1 (CSEF-1), bind to Enh4, which plays an essential role in enhancer function. In this study, we demonstrate that TEF-1 and CSEF-1 bind specifically to all the other GT-IIC- and SphI/II-like elements within CSEn with a broad range of binding affinities that vary between 0.005 and 0.15 that of Enh4. Each individual concatenated enhanson was able to stimulate hCS promoter activity in an orientation-independent manner in choriocarcinoma cells (BeWo) with an observed stimulation that was directly proportional to its relative binding affinity for TEF-1 and CSEF-1. These results indicate that CSEn function results from the cooperative interaction of TEF-1 and/or CSEF-1 binding to multiple, low-affinity GT-IIC- and SphI/II-like enhansons within the enhancer.

Identification of the human alpha6 integrin gene promoter.

The alpha6 integrin subunit couples with either the beta1 or the beta4 subunit to form a laminin receptor. alpha6 expression is cell-type-specific and generally is present at high levels in epithelial and endothelial cells. To study its gene regulation, we isolated a genomic clone containing the human alpha6 integrin gene promoter. It includes 3 kb of the upstream flanking region, the first exon (385 bp), and 9 kb of the first intron. The alpha6 promoter directs transcription initiation from a primary site 202 nucleotides from the translation initiation codon. Unlike most other integrin gene promoters, the alpha6 promoter has a TATA box (GATAAA), which is located 22 nucleotides upstream from the primary transcription initiation site. A 190-bp region upstream from the TATA box is highly rich (78%) in C and G nucleotides and contains several Sp1 and AP2 binding sequences. However, full promoter activity (in the presence of the SV40 enhancer) requires only 78 bp of this C/G-rich sequence upstream from the TATA box. Slightly upstream from the C/G-rich region are a steroid receptor binding homolog and an epithelial-cell-specific E-pal sequence. Another possible epithelial cell-specific binding sequence (Ker1) is found immediately downstream from the TATA box. Cell-type-specific activities of the promoter paralleled the alpha6 mRNA levels in four tested cell lines. In the presence of the SV40 enhancer, alpha6 promoter activity increased approximately four-fold in primary keratinocytes and in HT1080 fibrosarcoma cells and 30-fold in T47D breast carcinoma cells, but remained undetectable in K562 leukemia cells. Genomic analysis that compared alpha6-expressing with non-alpha6-expressing cells suggested that DNA methylation is not involved in the silencing of the alpha6 gene in alpha6-negative cells. DNase I footprint analysis confirmed the binding of Sp1 and AP2 to their cognate sequences. A nuclear extract of high-alpha6-expressing HBL-100 cells also produced significant binding to these sites, suggesting that the two transcription factors are probably involved in the positive regulation of the alpha6 promoter.

DNA elements recognizing NF-Y and Sp1 regulate the human multidrug-resistance gene promoter.

Regulation of the human multidrug resistance gene (hMDR1) was studied by mapping DNA elements in the proximal promoter necessary for efficient transcription. Transient transfection analysis in tumor cell lines (HCT116, HepG2, and Saos2) of promoter deletions identified several regulatory domains. These cell lines expressed hMDR1 mRNA. Removal of an element between +25 and +158 reduced promoter activity by 2-3-fold, whereas deletion of sequences from approximately -5000 to -138 base pairs gave a approximately 2-fold increase. The activity of the hMDR1 promoter (-137 to +25) was comparable in activity to the SV40 early promoter and enhancer combination. Deletion of the hMDR1 promoter between -86 and -44 reduced activity by 5-10-fold, identifying an important regulatory region. This minimal region (-88 to -37) activated transcription when inserted upstream of a synthetic promoter, suggesting that it acts independently of other regulatory sequences. Two DNA elements within 85 base pairs of the transcriptional start site were required to confer efficient gene expression. A double-point mutation in the Y box (inverted CCAAT box) between -70 and -80 reduced activity of the promoter by 5-10-fold, and a single-point mutation at -52 within a GC-rich element reduced activity by 3-fold. Thus, both the Y-box and GC elements must each remain intact for optimal promoter activity. DNA-binding analyses suggest that the transcription factor NF-Y, but not YB-1 or c/EBP, is most likely responsible for controlling the activity of the Y-box element in these tumor cell lines. DNA-binding analyses also suggest that Sp1, alone or in combination with other nuclear factors, likely controls the activity of the GC element.

Regulation of expression of the human heme oxygenase-1 gene in transfected chick embryo liver cell cultures

Induction of heme oxygenase (HO) has been proposed as a protective cellular mechanism against oxidative damage. In previous work (Tyrrell et al., Carcinogenesis [1993] 14, 761–765), portions of the 5′ promoter region of the human HO-1 gene linked to the reporter gene chloramphenicol acetyl transferase (CAT), had been transiently expressed in HeLa cells. To extend the study of human HO gene expression into primary liver cells, these reporter gene fusion constructs, containing 121 or 1416 base pairs of the untranscribed 5′-upstream sequences of the human HO-1 gene, were used along with pSV β-Gal plasmid to dually transfect primary cultures of chick embryo liver cells (CELC). The transfected cells were treated with selected metals, heme, phorbol ester, and chemical agents that produce oxidative stress (H 2O 2 or sodium arsenite). Reporter gene activities were measured 18–20 h later. Our major findings are: (1) these HO-CAT constructs were expressed in CELC; (2) unlike HeLa cells, the expression of CAT was detected in CELC without the need for the SV40 enhancer; (3) sodium arsenite and cobalt chloride induced the expression of the HO-CAT constructs whereas heme had no effect on or decreased CAT expression for all of the transfected constructs; (4) study of endogenous chick HO-1 gene expression in CELC showed that HO-1 responded to sodium arsenite treatment in a dose-dependent fashion, and the response was rapid and transient. We conclude that, in chick liver cell cultures, induction of the HO-1 gene by heme is fundamentally different from that produced by transition metals or sodium arsenite. Furthermore, the results suggest that expression of the HO-1 gene is highly conserved across species. © 1997 Elsevier Science B.V. All rights reserved.

Analysis of genes for human snRNP Sm-D1 protein and identification of the promoter sequence which shows segmental homology to the promoters of Sm-E and U1 snRNA genes

The Sm core proteins of U1, U2, U4/U6 and U5 snRNPs include B(B1), B′(B2), N(B3), D1, D2, D3, E, F and G polypeptides. We have isolated genomic clones encoding the Sm-D1 protein using the Sm-D1 cDNA as probe. Southern blotting and DNA sequencing analysis of these clones revealed the presence of an Sm-D1 multigene family in the human genome. Three gene members have been identified. Two of the genes are without introns and contain mutations compared to the cDNA sequence. They appear to be processed pseudogenes. The third gene, termed SNRPD1, shares 100% identity to the cDNA sequence including both 5′- and 3′-untranslated regions ( UTR); it contains three introns. Analysis of the 5′-flanking region of the SNRPD1 gene revealed promoter activity, suggesting this is the functional gene that encodes the Sm-D1 protein. The promoter activity was localized in a 0.38 kb PstI fragment using CAT reporter gene fusion assays. Addition of an SV40 enhancer element did not enhance the transcription directed by that fragment. Sequence comparison of the 0.38 kb promoter sequence with the promoters of the Sm-E gene and U1 snRNA genes revealed several homologous motifs, suggesting that genes encoding the snRNP components may be coordinately regulated.

Functional characterization of a chicken major histocompatibility complex class II B gene promoter.

A 0.7 kilobase (kb) DNA fragment from the 5' flanking region of a chicken major histocompatibility complex (MHC) class II B gene was cloned into chloramphenicol acetyltransferase (CAT) reporter vectors and was transfected into a chicken macrophage cell line that expresses a low level of MHC class II antigens. Positive orientation-dependent promoter activity of the chicken DNA was evident in a reporter construct containing an SV40 enhancer. Deletion analysis of this 0.7 kb DNA fragment revealed a short fragment in the 3' end that was crucial for the promoter function and negative regulatory elements (NRE) located further upstream. The conserved MHC class II X and Y boxes did not have a significant effect on promoter activity. Sequence analysis of the 0.7 kb class II B gene upstream region suggests possible involvement of interferon (IFN), E twenty-six specific (ETS)-related proteins, and other factors in regulating this promoter. A chicken T-cell line culture supernatant increased surface expression of MHC class II antigens, as well as class II promoter activity, in this macrophage cell line. This first functional characterization of a chicken MHC class II B gene promoter will aid in understanding the regulatory mechanisms that control the expression of these genes.

Sperm as a carrier to introduce an exogenous DNA fragment into the oocyte of Japanese abalone (Haliotis divorsicolor suportexta).

We investigated gene transfer in abalone via electroporated sperm. The mobility of sperm electroporated either in seawater or in marine invertebrate physiological solution was as good as that of the control group. The fertilization rate reached as high as 94.7-99.6% (93.0-99.7% for the control group) when 200 eggs were fertilized by 10(6) or 10(7) sperm treated with electroporation at 10 kV and 2(7) pulses for six cycles. Moreover, the fertilization rate of sperm electroporated in the presence of foreign DNA (opAFP-2000CAT) ranging from 0.1 to 3.2 micrograms and at voltages ranging from 2 to 10 kV, at 2(7) or 2(11) pulses for six or 12 cycles showed no differences from the control sperm. After DNase digestion, the genome of the electroporated sperm was analysed by polymerase chain reaction, and it was shown that a 138-bp product was amplified, corresponding to the transgene's amplification product. Southern blotting also showed that a positive band located at the same position as that of opAFP-2000CAT was found in the electroporated sperm after DNase treatment. Analysis by PCR of the genome isolated from a trochophore-stage abalone larva, derived from sperm electroporated with 3.2 micrograms opAFP-2000CAT, showed the existence of foreign DNA in 13 out of 20 examined samples (65%). The integration of the transferred DNA into the genome of transgenic abalone was also shown by Southern blot analysis. Furthermore, CAT activity was positive for the experimental larvae, but the level of CAT expression was lower than that of larvae derived from sperm electroporated with pCAT-Control vector, driven by SV40 promoter and enhancer sequences. These results demonstrate the potential for the use of sperm as mass gene transfer strategy in marine mollusks such as abalone.

LTR retrotransposons and the evolution of eukaryotic enhancers.

Since LTR retrotransposons and retroviruses are especially prone to regional duplications and recombination events, these viral-like systems may be especially conducive to the evolution of closely spaced combinatorial regulatory motifs. Using the Drosophila copia LTR retrotransposon as a model, we show that a regulatory region contained within the element's untranslated leader region (ULR) consists of multiple copies of an 8 bp motif (TTGTGAAA) with similarity to the core sequence of the SV40 enhancer. Naturally occurring variation in the number of these motifs is correlated with the enhancer strength of the ULR. Our results indicate that inter-element selection may favor the evolution of more active enhancers within permissive genetic backgrounds. We propose that LTR retroelements and perhaps other retrotransposons constitute drive mechanisms for the evolution of eukaryotic enhancers which can be subsequently distributed throughout host genomes to play a role in regulatory evolution.

Bent DNA in the Human Adenovirus Type 2 E1A Enhancer Is an Architectural Element for Transcription Stimulation

Bent DNA in the Human Adenovirus Type 2 E1A Enhancer Is an Architectural Element for Transcription Stimulation

Protein-DNA Interactions at a Drug-responsive Element of the Human Apolipoprotein A-I Gene

Previously, we demonstrated that when two human hepatoma cell lines, Hep3B and HepG2, were exposed to gemfibrozil, a hypolipidemic drug, a 2-fold induction in apolipoprotein A-I (apoA-I) mRNA levels resulted. To determine if mRNA stabilization was responsible for the changes in apoA-I mRNA levels, the half-lives for apoA-I mRNA were measured in the presence of actinomycin D with and without gemfibrozil. These experiments revealed no differences in stability. However, nuclear run-off assays indicated that the transcription rate of the apoA-I gene was increased 2-fold in gemfibrozil-treated cells. Transient transfection experiments also indicated that the induction of apoA-I mRNA level in response to gemfibrozil is mediated at the transcriptional level. We have identified two copies of the "drug-responsive element" (DRE) in the apoA-I promoter region that may be responsible for the increase in apoA-I transcriptional activity by gemfibrozil. Using gel mobility shift assays with a synthetic DRE oligonucleotide, we have demonstrated that exposure of Hep3B and HepG2 cells to gemfibrozil resulted in strong induction of a protein-DNA complex. The formation of this complex is highly sequence-specific as indicated by the DNA competition experiments. The drug-inducible nuclear proteins bind to the DRE of the human apoA-I gene with an apparent Kd of 4.1 nM. Methylation interference experiments have localized the contact sites of nuclear factors to the DRE region. Southwestern blot analyses have identified two groups of drug-inducible nuclear proteins with molecular masses of approximately 30 and 15 kDa. When a copy of synthetic DRE oligonucleotide was inserted upstream of the thymidine kinase promoter and luciferase reporter construct, a significant 2-fold induction in luciferase activity was observed in the presence of gemfibrozil following transient transfection of two human hepatoma cell lines, HepG2 and Hep3B. However, a plasmid containing one copy of mutated apoA-I-DRE oligomer did not confer responsiveness to gemfibrozil treatment. Furthermore, pGL2 (apoA-I -250 mutant DRE), which carried an internal mutation of the DRE in the human apoA-I proximal promoter region, showed no increase in luciferase activity in response to gemfibrozil. These results implicate protein-DNA interactions at the DRE region in the transcriptional induction of human apoA-I gene expression by gemfibrozil.

A Novel Family of Developmentally Regulated Mammalian Transcription Factors Containing the TEA/ATTS DNA Binding Domain

We describe the molecular cloning of two novel human and murine transcription factors containing the TEA/ATTS DNA binding domain and related to transcriptional enhancer factor-1 (TEF-1). These factors bind to the consensus TEA/ATTS cognate binding site exemplified by the GT-IIC and Sph enhansons of the SV40 enhancer but differ in their ability to bind cooperatively to tandemly repeated sites. The human TEFs are differentially expressed in cultured cell lines and the mouse (m)TEFs are differentially expressed in embryonic and extra-embryonic tissues in early post-implantation embryos. Strikingly, at later stages of embryogenesis, mTEF-3 is specifically expressed in skeletal muscle precursors, whereas mTEF-1 is expressed not only in developing skeletal muscle but also in the myocardium. Together with previous data, these results point to important, partially redundant, roles for these TEF proteins in myogenesis and cardiogenesis. In addition, mTEF-1 is strongly coexpressed with mTEF-4 in mitotic neuroblasts, while accentuated mTEF-4 expression is also observed in the gut and the nephrogenic region of the kidney. These observations suggest additional roles for the TEF proteins in central nervous system development and organogenesis.

Identification and characterization of two promoters of rat kallikrein-binding protein gene

Rat kallikrein-binding protein (RKBP) is a serine proteinase inhibitor (serpin) which binds to and inhibits tissue kallikrein activity [1,2]. In this study, we have sequenced and identified two promoter regions of the RKBP gene ( RKBP). One promoter is located in the 5′ flanking region (P 2) of the gene and the other is located in the first intron (P 2). Both promoters contain a consensus TATA and CAAT box. These RKBP promoters were fused with a chloramphenicol acetyltransferase (CAT) reporter gene and their promoter activities were determined by measuring CAT levels using a specific ELISA. The P 1 promoter exhibited high promoter activities in Hep3B hepatoma cells but not in La-fibroblastoma cells, indicating its tissue-specificity. By deletion analysis, we have identified a negative regulatory element of the P, promoter between −739 and −472, and defined a minimal sequence between − 183 and −2 for maintaining the intact promoter activity. The P 2 promoter showed a strong activity only when linked to an SV40 enhancer. Activity of the P 1 promoter can be induced by growth hormone in Hep3B cells. Gel retardation assay has identified 5 DNA fragments which were bound by nuclear proteins from rat liver. Two DNA fragments are in the 5′ flanking region, one contains a putative glucocorticoid and growth hormone response element and the other one contains a CAAT box and two putative AP-1 binding sites. The remaining three are in the first intron and contain a putative thyroid hormone response element, a putative GATA site and three consensus CAAT boxes, respectively. Nuclear proteins from the kidney showed that spontaneously hypertensive rats (SHR) have a distinct trans-acting factor which binds with the DNA fragment containing the glucocorticoid and growth hormone response elements, as compared with normotensive rats. This result indicates that different trans-acting factors in the kidney of SHR may contribute to the decreased RKBP expression in these hypertensive rats.

Identification of a Murine TEF-1-related Gene Expressed after Mitogenic Stimulation of Quiescent Fibroblasts and during Myogenic Differentiation

Fibroblast growth factor (FGF)-1 binding to cell surface receptors stimulates an intracellular signaling pathway that ultimately promotes the transcriptional activation of specific genes. We have used a mRNA differential display method to identify FGF-1-inducible genes in mouse NIH 3T3 fibroblasts. Here, we report that one of these genes, FGF-regulated (FR)-19, is predicted to encode a member of the transcriptional enhancer factor (TEF)-1 family of structurally related DNA-binding proteins. Specifically, the deduced FR-19 amino acid sequence has approximately89, 77, and 68% overall identity to chicken TEF-1A, mouse TEF-1, and mouse embryonic TEA domain-containing factor, respectively. Gel mobility shift experiments indicate that FR-19, like TEF-1, can bind the GT-IIC motif found in the SV40 enhancer. The FR-19 gene maps in the distal region of mouse chromosome 6, and analysis of several FR-19 cDNA clones indicates that at least two FR-19 isoforms may be expressed from this locus. FGF-1 induction of FR-19 mRNA expression in mouse fibroblasts is first detectable at 4 h after FGF-1 addition and is dependent on de novo RNA and protein synthesis. FGF-2, calf serum, platelet-derived growth factor-BB, and phorbol 12-myristate 13-acetate can also induce FR-19 mRNA levels. We have also found that FR-19 mRNA expression increases during mouse C2C12 myoblast differentiation in vitro. The FR-19 gene is expressed in vivo in a tissue-specific manner, with a relatively high level detected in lung. These results indicate that increased expression of a TEF-1-related protein may be important for both mitogen-stimulated fibroblast proliferation and skeletal muscle cell differentiation.

Activation of Epidermal Growth Factor Receptor Gene Transcription by Phorbol 12-Myristate 13-Acetate Is Mediated by Activator Protein 2

The response of the epidermal growth factor (EGF) receptor gene to phorbol 12-myristate 13-acetate (PMA) was analyzed using nuclei and nuclear extracts prepared from PMA-treated KB cells. Transient transfection assays and nuclear run-off experiments showed that PMA increased EGF receptor gene transcription. Cell-free transcription with promoter mutants revealed that the region of the promoter containing nucleotides -150 to -16 was sufficient for PMA inducibility. A promoter fragment containing nucleotides -167 to -105 showed increased binding of a factor present in extracts prepared from PMA-treated cells. When this factor was partially purified by column chromatography, it showed specific PMA-dependent binding to an EGF receptor promoter fragment. This binding was competed by an SV40 fragment containing binding sites for Sp1, AP1, and AP2. Purified AP2 was used in DNase I footprinting experiments to show that this factor can bind to the EGF receptor promoter. Oligonucleotides corresponding to the AP2 binding sites found in the EGF receptor promoter showed the ability to bind AP2 and compete for the binding of a factor induced by PMA treatment. The addition of AP2 to nuclear extract resulted in increased transcription from the EGF receptor promoter. These results demonstrate that AP2 can activate EGF receptor gene expression and may mediate the PMA response of this gene.

Cell-type-specific modulation of PDGF-B regulatory elements via viral enhancer competition: a caveat for the use of reference plasmids in transient transfection assays

The human platelet-derived growth factor-B (PDGF-B) gene has been shown to display a wide range of levels of mRNA transcription in a variety of cell types. Functional analyses of PDGF-B gene expression have begun to reveal intricate, cell-type-specific regulatory mechanisms involving multiple control elements. We have previously isolated and characterised several elements involved in the control of human PDGF-B gene expression in the JEG-3 choriocarcinoma cell line and in the breast cancerderived cell line, ZR-75. Assessment of the positive or negative regulatory contributions of these elements was carried out using transient transfection assays. Such studies routinely require the inclusion of a reference plasmid in order to determine transfection efficiency. Here we show that competition for regulatory factors occurs in transfected cells between vital enhancers and elements regulating PDGF-B gene transcription. A frequently used reference plasmid which utilises the SV40 promoter and enhancer region to drive expression of a β-galactosidase reporter gene was found to severely repress the activity of a co-transfected reporter construct containing the PDGF-B promoter and its intronic enhancer in JEG-3 cells. This competition was localised to the enhancer region of the SV40 regulatory sequences and surprisingly, the effect was reversed in ZR-75 cells; where increasing the amount of reference plasmid strongly stimulated the activity of the PDGF-B construct. These results imply that the same intronic region which functions equally well as an enhancer in two distinct cell-types, may operate in response to different transcription factor complements. Furthermore, this data demonstrates that the choice of reference plasmid and its quantitative use can be a crucial factor when examining putative regulatory elements by transient transfection methods.

Interaction of Oct-1 with TFIIB: IMPLICATIONS FOR A NOVEL RESPONSE ELICITED THROUGH THE PROXIMAL OCTAMER SITE OF THE LIPOPROTEIN LIPASE PROMOTER

The ubiquitous human POU domain protein, Oct-1, and the related B-cell protein, Oct-2, regulate transcription from a variety of eukaryotic genes by binding to a common cis-acting octamer element, 5'-ATTTGCAT-3'. The binding of Oct-1 and Oct-2 to the functionally important lipoprotein lipase (LPL) promoter octamer site was stimulated by the general transcription factor, TFIIB. Comparative analysis of the LPL, histone H2B (H2B), and herpes simplex virus ICPO gene promoter octamer sites revealed that nucleotide sequences within and flanking the octamer sequence determined the degree of TFIIB-mediated stimulation of Oct-1 DNA binding. TFIIB was found to decrease the rate of dissociation of Oct-1 from the LPL octamer site, whereas it increased the rate of association, as well as decreased the rate of dissociation, of Oct-1 from the H2B octamer site. A monoclonal antibody against TFIIB immunoprecipitated a ternary complex containing TFIIB, Oct-1, and the LPL and H2B octamer binding sites. TFIIB did not alter the DNase I footprints generated by Oct-1 on the LPL and H2B promoters. However, Oct-1 on the TATA-binding protein and TFIIB from footprinting the perfect TATA box sequence located 5' of the LPL, NF-Y binding site. In transfection experiments, transcription from the reporters containing the LPL octamer, and either the SV40 or the yeast transcription factor GAL4-dependent enhancers, initiated at a precise position within the octamer sequence. Transcription from reporters containing the H2B octamer and the SV40 enhancer initiated at several positions within and flanking the octamer site, whereas transcription initiated at a precise position within the octamer from reporters with both the H2B octamer and the GAL4-dependent enhancer. These results suggest that octamers and their flanking sequences play an important role in positioning the site of transcription initiation, and that this could be a function of the interaction of Oct-1 with TFIIB.

Cloning of the murine CD5 promoter and its tissue-specific regulation.

A genomic clone containing 1700 bp of the 5'-flanking region and first exon of the murine CD5 gene was isolated by screening a NIH 3T3 fibroblast genomic library with the previously characterized murine CD5 cDNA. The CD5 5'-flanking region lacks a consensus TATA box but contains Sp1, AP-1, IgH muE2, SV40 enhancer core, CCAAT, TCF2 alpha/PEA3/ets, and C/EBP motifs. Transcription is initiated from multiple sites upstream of an Inr-like sequence. When linked to the CAT reporter gene, the CD5 5'-flanking region was an active promoter in transient transfection assays using the EL4 (T), PD36 (pre-B) and M12 (B) cell lines, but was inactive in NIH 3T3 fibroblasts. This pattern of lymphoid-specific expression reflects the pattern of in vivo CD5 expression. Successive 5' to 3' deletions of the CD5 promoter/CAT reporter construct were transfected into T and B cells revealing a succession of positive and negative regulatory elements until promoter activity was eliminated at position -27. This result identified sequences from -125 to -27 (which contain the Inr, IgH muE2, SV40 enhancer core, and AP-1 sites) as sufficient for tissue-specific promotion. Isolation and characterization of the CD5 promoter represents an initial step in elucidating the control over tissue-specific expression of the murine CD5 gene.

Cloning of an SNF2/SWI2-related Protein That Binds Specifically to the SPH Motifs of the SV40 Enhancer and to the HIV-1 Promoter

We have isolated a human cDNA clone encoding HIP116, a protein that binds to the SPH repeats of the SV40 enhancer and to the TATA/inhibitor region of the human immunodeficiency virus (HIV)-1 promoter. The predicted HIP116 protein is related to the yeast SNF2/SWI2 transcription factor and to other members of this extended family and contains seven domains similar to those found in the vaccinia NTP1 ATPase. Interestingly, HIP116 also contains a C3HC4 zinc-binding motif (RING finger) interspersed between the ATPase motifs in an arrangement similar to that found in the yeast RAD5 and RAD16 proteins. The HIP116 amino terminus is unique among the members of this family, and houses a specific DNA-binding domain. Antiserum raised against HIP116 recognizes a 116-kDa nuclear protein in Western blots and specifically supershifts SV40 and HIV-1 protein-DNA complexes in gel shift experiments. The binding site for HIP116 on the SV40 enhancer directly overlaps the site for TEF-1, and like TEF-1, binding of HIP116 to the SV40 enhancer is destroyed by mutations that inhibit SPH enhancer activity in vivo. Purified fractions of HIP116 display strong ATPase activity that is preferentially stimulated by SPH DNA and can be inhibited specifically by antibodies to HIP116. These findings suggest that HIP116 might affect transcription, directly or indirectly, by acting as a DNA binding site-specific ATPase.

Characterization of the promoter region of the human MDR3 P-glycoprotein gene

The human MDR3 (or MDR2) P-glycoprotein is probably involved in the transport of phospholipids from liver hepatocytes into bile (Smit et al. (1993) Cell 75, 451–462). In accordance with this function, MDR3 is highly expressed in human liver, but lower mRNA levels were also found in adrenal, heart, muscle and cells of the B-cell compartment. We have cloned and analyzed the MDR3 promoter region. It is GC-rich, and contains neither a TATA nor a CART box, but it does contain multiple putative SP1 binding sites, features also found in so-called housekeeping genes. RNase protection and primer extension analyses indicate that the MDR3 gene has multiple transcription start sites in a GC-rich region with considerable homology to the putative mouse mdr2 promoter. A 3 kb genomic fragment containing the MDR3 start sites directs transcription of a chloramphenicol acetyltransferase (CAT) reporter gene upon transient transfection in the human hepatoma cell line HepG2. This transcription is orientation dependent, and stimulated by a SV40 enhancer, indicating that the 3 kb insert contains the core promoter elements of the MDR3 gene. The promoter region contains several consensus sequences where known or putative liver-specific (C/EBP, HNF5) or lymphoid specific (Pu.1, ets-1) transcription factors may bind.