Major Promoter Elements Research Articles

Evolution of the interferon τ genes and their promoters, and maternal–trophoblast interactions in control of their expression

It is well established that the interferon τ (IFN-τ) family of proteins play a major role in preventing the regression of the corpus luteum during early pregnancy in ruminants, such as cattle, sheep and goats, but not in other mammals. These interferons, which are structurally and functionally related to type I interferon, such as IFN-α and -ω, arose from a duplication of an IFN-ω gene approximately 36 million years ago. The IFN-τ genes have continued to duplicate since that time and have acquired the ability to be transcribed uniquely in the trophectoderm. Low expression is first detectable at the blastocyst stage, but massive transcriptional upregulation occurs a few days later during the initial stages of conceptus elongation. Expression is finally terminated upon trophectoderm attachment to uterine endometrium. The major promoter element that controls expression is an Ets-2/AP-1 enhancer element. Growth factors and cytokines released by the maternal endometrium that, possibly in response to progesterone, act through Ras and the mitogen-activated protein kinase (MAP-kinase) signal transduction pathway have been implicated in controlling IFN-τ gene transcription by activating Ets-2. This timely expression of IFN-τ is not only required to rescue the corpus luteum of pregnancy but may also be an indicator of conceptus fitness, thereby serving as a critical factor that dictates the continuation of pregnancy in ruminants.

CpG islands and GC content dictate nucleosome depletion in a transcription-independent manner at mammalian promoters

One clear hallmark of mammalian promoters is the presence of CpG islands (CGIs) at more than two-thirds of genes, whereas TATA boxes are only present at a minority of promoters. Using genome-wide approaches, we show that GC content and CGIs are major promoter elements in mammalian cells, able to govern open chromatin conformation and support paused transcription. First, we define three classes of promoters with distinct transcriptional directionality and pausing properties that correlate with their GC content. We further analyze the direct influence of GC content on nucleosome positioning and depletion and show that CpG content and CGI width correlate with nucleosome depletion both in vivo and in vitro. We also show that transcription is not essential for nucleosome exclusion but influences both a weak +1 and a well-positioned nucleosome at CGI borders. Altogether our data support the idea that CGIs have become an essential feature of promoter structure defining novel regulatory properties in mammals.

Promoter methylation confers kidney‐specific expression of the Klotho gene

The aging suppressor geneKlotho is predominantly expressed in the kidney irrespective of species. Because Klotho protein is an essential component of an endocrine axis that regulates renal phosphate handling, the kidney-specific expression is biologically relevant; however, little is known about its underlying mechanisms. Here we provide in vitro and in vivo evidence indicating that promoter methylation restricts the expression of the Klotho gene in the kidney. Based on evolutionary conservation and histone methylation patterns, the region up to -1200 bp was defined as a major promoter element of the human Klotho gene. This region displayed promoter activity equally in Klotho-expressing and -nonexpressing cells in transient reporter assays, but the activity was reduced to ∼20% when the constructs were integrated into the chromatin in the latter. Both endogenous and transfected Klotho promoters were 30-40% methylated in Klotho-nonexpressing cells, but unmethylated in Klotho-expressing renal tubular cells. DNA demethylating agents increased Klotho expression 1.5- to 3.0-fold in nonexpressing cells and restored the activity of silenced reporter constructs. Finally, we demonstrated that a severe hypomorphic allele of Klotho had aberrant CpG methylation in kl/kl mice. These findings might be useful in therapeutic intervention for accelerated aging and several complications caused by Klotho down-regulation.

Cloning and biological characterization of buffalo ( Bubalus bubalis) interferon-γ

Interferon-γ, a major immunomodulatory cytokine, of Indian water buffalo ( Bubalus bubalis) was characterized at molecular level. Complementary DNA and essential promoter region were cloned and sequenced, and functional recombinant protein was expressed in bacterial system. The cDNA has 97.8% nucleotide identity with 11-nucleotide and four-amino acid variations, and the essential promoter region has 98.4% identity with five-nucleotide variations and a four-nucleotide deletion in comparison with the corresponding bovine sequences. All the major promoter elements such as NF IL-2 like motif, cyclosporin sensitive binding element and GATA motif are strictly conserved. Recombinant buffalo-IFN-γ expressed in bacterial system reacted with an anti-bovine-IFN-γ monoclonal antibody in Western blot and showed antiviral activity against buffalo pox virus in cultured Madin-Darby bovine kidney (MDBK) cells by inhibiting virus induced cytopathic effect. The study shows high level sequence similarity of IFN-γ among ruminants. In view of the immunomodulatory and antiviral activities of IFN-γ, this molecule will be useful in better understanding of the immune system of water buffaloes.

Analysis of the human hexokinase II promoter in vivo: lack of insulin response within 4.0 kb

To locate regulatory element(s) that mediate(s) the effect of insulin on hexokinase II (HKII) gene transcription, we have generated several transgenic mouse lines harboring 97, 254, 505, 819 or 4077 bp of the proximal promoter of the human HKII gene driving the expression of a luciferase reporter gene. Luciferase activities indicate that major promoter elements responsible for the basal activity and tissue-specificity of the human HKII gene expression are located within the 505-bp segment of the promoter. To induce the promoter constructs by endogenous insulin released from the pancreatic β-cells, transgenic mice were given repeated intraperitoneal injections of d-glucose. Significant induction of luciferase activity was not observed in any of the transgenic mouse lines even though the endogenous HKII mRNA was induced 2.7-fold upon treatment. Thus, our results suggest a lack of insulin response in the 4.0-kb region of the proximal promoter of the human HKII gene in mice.

Interaction of proteins with promoter elements of the human U2 snRNA genes in vivo

The multicopy human U2 small nuclear (sn)RNA genes are transcribed by RNA polymerase (pol) II and contain two major promoter elements upstream of the transcription start site: an essential proximal sequence element (PSE) at around −55 and a distal sequence element (DSE) at around −220. We have carried out an in vivo footprinting analysis on these genes, and the results suggest that most, if not all, of the U2 gene promoters are bound by factors in interphase. Both the DSE and the PSE are protected from digestion, and the pattern of methylation protection over the DSE is virtually identical to that obtained in vitro using nuclear extract. Our results also indicate that the DNA between the PSE and the transcription start site is distorted and that proteins interact with the promoter between −20 and −33. Mutation of this sequence affects both the accuracy of initiation and polymerase specificity, underlining the importance of this region in U2 gene expression. We have also analysed the pattern of protection over the DSE and PSE of the U2 genes in mitotic cells. The degree of protection over all promoter elements is drastically reduced, suggesting that loss of DNA binding factors from the promoter plays a role in the shutdown of U2 gene transcription in mitosis.

The Eukaryotic Promoter Database, EPD: new entry types and links to gene expression data.

The Eukaryotic Promoter Database (EPD) is an annotated, non-redundant collection of eukaryotic Pol II promoters, for which the transcription start site has been determined experimentally. Access to promoter sequences is provided by pointers to positions in nucleotide sequence entries. The annotation part of an entry includes a description of the initiation site mapping data, exhaustive cross-references to the EMBL nucleotide sequence database, SWISS-PROT, TRANSFAC and other databases, as well as bibliographic references. EPD is structured in a way that facilitates dynamic extraction of biologically meaningful promoter subsets for comparative sequence analysis. World Wide Web-based interfaces have been developed which enable the user to view EPD entries in different formats, to select and extract promoter sequences according to a variety of criteria, and to navigate to related databases exploiting different cross-references. The EPD web site also features yearly updated base frequency matrices for major eukaryotic promoter elements. EPD can be accessed at http://www.epd.isb-sib.ch.

Transcriptional activity of human papillomavirus type 16 variants having deletions in the long control region

Transcription of the E6 and E7 viral oncogenes of human papillomavirus (HPV) type 16 is regulated by the P97 major early promoter, and enhancer and silencer elements found in the long control region (LCR). In this study, we tested the transcriptional activity of natural HPV 16 variants having long deletions in the LCR. The HPV 16 LCR regions were amplified from invasive cervical cancer specimens, and cloned into the reporter vector pALuc. Transcriptional activity of the different clones was measured by luciferase test after transient transfection into HeLa cells. The deletions found in the LCR encompassed parts of the enhancer and either the YY1-specific silencer alone or together with the CDP-specific silencer. The transcriptional activity of these deletion variants were usually reduced compared with that of the corresponding full-length clones. However, a deletion variant lacking the whole enhancer and both silencer regions retained substantial enhancer activity on the P97 promoter. These results point to the existence of a novel context-dependent enhancer element in the 5′ LCR of HPV 16.

The human gamma-globin TATA and CACCC elements have key, distinct roles in suppressing beta-globin gene expression in embryonic/fetal development.

The competition model of globin gene regulation states that the gamma-globin gene precludes expression of the beta-globin gene in early development by competing for the enhancing activity of the locus control region. The gamma-globin gene with a -161 promoter is sufficient for suppressing beta-globin gene expression, and the gamma-globin TATA and CACCC elements are necessary for this effect. In this work, stable transfection and transgenic mouse assays have been performed with constructs containing HS3 and HS2 from the locus control region, the gamma-globin gene with promoter mutation(s), and the beta-globin gene. The data indicate that the gamma-globin TATA and CACCC elements together have at least an additive effect on the beta/gamma-globin mRNA ratio in early erythroid cells, suggesting that the elements work coordinately to suppress beta-globin gene expression. The TATA and CACCC are the major gamma-globin promoter elements responsible for this effect. Transgenic mouse experiments indicate that the gamma-globin TATA element plays a role in gamma-globin expression and beta-globin suppression in the embryo and fetus; in contrast, the CACCC element has a stage-specific effect in the fetus. The results suggest that, as is true for the erythroid Krüppel-like factor (EKLF) and the beta-globin promoter CACCC, a protein(s) binds to the gamma-globin CACCC element to coordinate stage-specific gene expression.

Identification and characterization of the potential promoter regions of 1031 kinds of human genes.

To understand the mechanism of transcriptional regulation, it is essential to identify and characterize the promoter, which is located proximal to the mRNA start site. To identify the promoters from the large volumes of genomic sequences, we used mRNA start sites determined by a large-scale sequencing of the cDNA libraries constructed by the "oligo-capping" method. We aligned the mRNA start sites with the genomic sequences and retrieved adjacent sequences as potential promoter regions (PPRs) for 1031 genes. The PPR sequences were searched to determine the frequencies of major promoter elements. Among 1031 PPRs, 329 (32%) contained TATA boxes, 872 (85%) contained initiators, 999 (97%) contained GC box, and 663 (64%) contained CAAT box. Furthermore, 493 (48%) PPRs were located in CpG islands. This frequency of CpG islands was reduced in TATA(+)/Inr(+) PPRs and in the PPRs of ubiquitously expressed genes. In the PPRs of the CGM2 gene, the DRA gene, and the TM30pl genes, which showed highly colon specific expression patterns, the consensus sequences of E boxes were commonly observed. The PPRs were also useful for exploring promoter SNPs.

Identification and Characterization of the Potential Promoter Regions of 1031 Kinds of Human Genes

To understand the mechanism of transcriptional regulation, it is essential to identify and characterize the promoter, which is located proximal to the mRNA start site. To identify the promoters from the large volumes of genomic sequences, we used mRNA start sites determined by a large-scale sequencing of the cDNA libraries constructed by the “oligo-capping” method. We aligned the mRNA start sites with the genomic sequences and retrieved adjacent sequences as potential promoter regions (PPRs) for 1031 genes. The PPR sequences were searched to determine the frequencies of major promoter elements. Among 1031 PPRs, 329 (32%) contained TATA boxes, 872 (85%) contained initiators, 999 (97%) contained GC box, and 663 (64%) contained CAAT box. Furthermore, 493 (48%) PPRs were located in CpG islands. This frequency of CpG islands was reduced in TATA+/Inr+PPRs and in the PPRs of ubiquitously expressed genes. In the PPRs of the CGM2 gene, the DRA gene, and theTM30pl genes, which showed highly colon specific expression patterns, the consensus sequences of E boxes were commonly observed. The PPRs were also useful for exploring promoter SNPs.[The nucleotide sequences described in this paper have been deposited in the DDBJ, EMBL, and GenBank data libraries under accession nos.AU098358–AU100608.]

Transcriptional activation of cathepsin D gene expression by 17β-estradiol: mechanism of aryl hydrocarbon receptor-mediated inhibition

17β-estradiol (E2) induces cathepsin D gene expression in MCF-7 human breast cancer cells and this response is inhibited by aryl hydrocarbon receptor (AhR) agonists, such as 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). Analysis of the cathepsin D gene promoter initially identified a pentanucleotide GCGTG core dioxin responsive element (DRE) that blocked E2 action by inhibiting formation of a transcriptionally active estrogen receptor (ER)-Sp1 complex. A second functional downstream inhibitory DRE (iDRE2) (−130 to −126) has now been identified in the cathepsin D gene promoter and inhibition of E2-induced transactivation involves inhibitory AhR crosstalk with the E2-responsive adenovirus major late promoter element (MLPE) at −124 to −104 in the cathepsin D gene promoter. The MLPE site primarily binds USF1/USF2 and ERα, and gel mobility shift and DNA footprinting assays show that the AhR complex decreases binding of these transcription factors to the MLPE.

Novel transcriptional regulatory signals in the adeno-associated virus terminal repeat A/D junction element.

Adeno-associated virus (AAV) type 2 vectors transfer stable, long-term gene expression to diverse cell types in vivo. Many gene therapy applications require the control of long-term transgene expression, and AAV vectors, similar to other gene transfer systems, are being evaluated for delivery of regulated gene expression cassettes. Previously, we (R. P. Haberman, T. J. McCown, and R. J. Samulski, Gene Ther. 5:1604-1611, 1998) demonstrated the use of the tetracycline-responsive system for long-term regulated expression in rat brains. In that study, we also observed residual expression in the "off" state both in vitro and in vivo, suggesting that the human cytomegalovirus (CMV) major immediate-early minimal promoter or other cis-acting elements (AAV terminal repeats [TR]) were contributing to this activity. In the present study, we identify that the AAV TR, minus the tetracycline-responsive minimal CMV promoter, will initiate mRNA expression from vector templates. Using deletion analysis and specific PCR-derived TR reporter gene templates, we mapped this activity to a 37-nucleotide stretch in the A/D elements of the TR. Although the mRNA derived from the TR is generated from a non-TATA box element, the use of mutant templates failed to identify function of canonical initiator sequences as previously described. Finally, we demonstrated the presence of green fluorescent protein expression both in vitro and in vivo in brain by using recombinant virus carrying only the TR element. Since the AAV terminal repeat is a necessary component of all recombinant AAV vectors, this TR transcriptional activity may interfere with all regulated expression cassettes and may be a problem in the development of novel TR split gene vectors currently being considered for genes too large to be packaged.

The eukaryotic promoter database (EPD).

The Eukaryotic Promoter Database (EPD) is an annotated non-redundant collection of eukaryotic POL II promoters for which the transcription start site has been determined experimentally. Access to promoter sequences is provided by pointers to positions in nucleotide sequence entries. The annotation part of an entry includes a description of the initiation site mapping data, exhaustive cross-references to the EMBL nucleotide sequence database, SWISS-PROT, TRANSFAC and other databases, as well as bibliographic references. EPD is structured in a way that facilitates dynamic extraction of biologically meaningful promoter subsets for comparative sequence analysis. WWW-based interfaces have been developed that enable the user to view EPD entries in different formats, to select and extract promoter sequences according to a variety of criteria, and to navigate to related databases exploiting different cross-references. The EPD web site also features yearly updated base frequency matrices for major eukaryotic promoter elements. EPD can be accessed at http://www.epd.isb-sib.ch

Characterization of the Human Aldehyde Reductase Gene and Promoter

Aldehyde reductase (EC 1.1.1.2; AKR1A1) is involved in the reduction of biogenic and xenobiotic aldehydes and is present in virtually every tissue. To study the regulation of its expression, the human aldehyde reductase gene and promoter were cloned and characterized. The protein coding region consists of eight exons, with two additional upstream exons, separated by a large intron of 9.4 kb, that code for the 5′ untranslated region of the mRNA. Two mRNA transcripts that encode the same protein and that originate from alternative splicing were identified. The shorter transcript is the major form as shown by Northern blots and reverse transcription-PCR experiments. Northern blots of multiple tissues indicate that aldehyde reductase mRNA is present in all tissues examined and is most abundant in kidney, liver, and thyroid, which is consistent with the tissue enzyme distribution. The two mRNA transcripts do not exhibit differential tissue distribution. A construct containing a promoter region insert in a pGL3 vector drives transcription of a luciferase reporter gene and is 290-fold more active than a control vector without insert in transfected HepG2 cells. The activity of the full promoter construct is comparable to that of a pGL3 vector containing the SV40 promoter with an enhancer. The promoter does not contain a TATA box, but contains multiple GC-rich islands and exhibits bidirectional activity in transfection studies. The major active promoter element was localized by nested deletions and mutations to a DNA element (TGCAAT, −59 to −54) that presumptively binds the transcription factor CHOP [CAAT enhancer binding protein (C/EBP) homologous protein]. Comparison of the aldehyde reductase gene structure to all other characterized human genes of the aldo–keto reductase superfamily (aldose reductase, bile acid binder, and type I and type II 3α-hydroxysteroid dehydrogenases) indicates that it is more distantly related to these genes than they are among themselves.

Isolation and Characterization of Synechococcus PCC7942 Promoters: tRNApro Gene Functions as a Promoter

Promoter-active fragments of Synechococcus PCC7942 were isolated by transcriptional gene fusion to the promoterless beta-glucuronidase (GUS) gene of E. coli, which was used as a reporter gene. Several of the isolated promoter-active fragments expressed GUS activity in Synechococcus comparable to that of the lambdaPR promoter. Only 10% of the isolated promoter-active fragments also functioned in E. coli. The transcription initiation sites of the two promoter-active fragments, D13 and E3, were identified. The major transcription initiation sites of D13 and E3 in Synechococcus were located within the nucleotides TTTG and TTG respectively, which were identical to those corresponding to E. coli. The inferred -10 and -35 regions of D13 were TAAACT and TTGTAG respectively, which conformed to the E. coli sigma70 promoter. Immediately upstream of the E3 transcription initiation sites was the tRNApro (GGG) gene, which contained two regions exhibiting strong homology to the major promoter elements in eukaryotic tRNA genes, but did not contain the E. coli promoter element. Thus, the tRNApro gene can act as a promoter.

Upstream stimulatory factors mediate estrogen receptor activation of the cathepsin D promoter.

Overexpression of cathepsin D (CD), a ubiquitous lysosomal protease, is closely associated with a poor clinical outcome for patients with breast cancer. Estrogen greatly induces transcription of the CD gene in estrogen receptor (ER)-positive breast cancer cells. In this report, we transiently introduced a human CD promoter/chloramphenicol acetyltransferase reporter gene into human MCF-7 breast cancer cells to study the mechanisms by which the ER activates the promoter. Using an in vivo Exonuclease III footprinting assay, we found that estrogen stimulation of MCF-7 cells induced loading of a transcription factor(s) to a portion of the promoter (-124 to -104) that is homologous to the adenovirus major late promoter element. Subsequent gel mobility shift assays with a 21-bp CD -124/-104 probe and nuclear extracts prepared from naive and estrogen-stimulated cells detected a single sequence-specific protein-DNA complex. Southwestern and UV cross-linking experiments detected two proteins of 44 kDa and 43 kDa that were specifically bound to the 21-bp fragment of the promoter. Gel super-shift assays with upstream stimulatory factor 1 (USF-1) and USF-2 antibodies demonstrated that USF-1 and USF-2 bound to the E box probe. Sequence specific binding was abolished by a 2-bp change shown previously to prevent the binding of USF to the E box. Incorporation of a mutant E box into the wild-type CD promoter/chloramphenicol acetyltransferase gene abolished USF binding and reduced the levels of both basal and estrogen-stimulated transcription. These results suggest that the ER targeting of USF-1 and USF-2 is a critical step in hormone activation of CD gene transcription in human breast cancer cells.

Plasmids for the in vitro analysis of RNA polymerase II-dependent transcription based on a G-free template

Described are a series of plasmids containing combinations of adenovirus-2 major late promoter elements, including consensus TATA box and initiator, upstream of G-free transcription cassettes of various lengths. These provide an assortment of tools for investigating both basal and regulated transcription mechanisms by in vitro transcription methods.

The 5'-terminal 32 basepairs conserved between genome segments A and B contain a major promoter element of infectious bursal disease virus.

SummaryThe regions of the infectious bursal disease virus (IBDV) genome with regulatory function are not known. In the present study, progressively deleted lengths of the 5′ noncoding region of segment A were constructed in pGL3 vectors having SV40 enhancer or promoter, and a luciferase (LUC) reporter gene. Transient transfections of the constructs made in a promoter-less pGL3-Enhancer vector when transfected in Vero cells and the lysates assayed for LUC expression, allowed the localization of maximal activity to the 32-nucleotide stretch (precursor polyprotein ORF positions −131 to −100), which is highly conserved at the 5′ end of both genome segments. This fragment, when evaluated in parallel in an enhancer-less pGL3-Promoter vector demonstrated no activity. To determine if this region is recognized by IBDV replicative proteins, we engineered modifications in an enhancer-less pGL3-Promoter vector where the terminal 32-bp fragment, the full-length noncoding region, or the noncodig region with the 32-bp fragment deleted was positioned in either the plus-sense or the minus-sense orientation immediately downstream of the SV40 promoter and upstream of the LUC gene. Transfections of these constructs in IBDV-infected and uninfected Vero cells resulted in the endogenous generation of recombinant viral-LUC RNAs containing the 5′ terminal viral RNA sequences in either the plus-sense or the minus-sense orientation. LUC assays of the infected cell lysates showed up-regulated expression of LUC only with constructs containing the 32-bp fragment in the minus-sense orientation. Deletion of this 32-bp fragment abolished such LUC expression. We therefore conclude that the 5′-terminal 32 base pairs of genomic segment A contain a major promoter element in IBDV. In addition, our results show that IBDV replicative proteins recognize and transcribe single-stranded RNA in vivo.

The CREB-binding Protein (CBP) Cooperates with the Serum Response Factor for Transactivation of the c-fos Serum Response Element

The serum response element is one of the major promoter elements of the immediate early response to extracellular signals. The serum response element includes two main binding sites for proteins: the Ets box, which binds p62(TCF), and the CArG box, which binds p67(SRF). These two proteins are direct targets for signal transduction pathways; p62(TCF) is a nuclear end point of the Ras/mitogen-activated protein kinase pathway, and p67(SRF) is targeted by the Rho/Rac small G-proteins. The mechanism by which the signal is further transduced from the transcription factors to the basal transcriptional machinery is poorly understood. Recent data have suggested that the cAMP-responsive element-binding protein (CREB)-binding protein, a transcriptional adaptor involved in the transactivation through a wide variety of enhancer elements, participates in p62(TCF) activity. We here show that the CREB-binding protein also cooperates in the process of transactivation by p67(SRF). Cotransfections of expression vectors for the CREB-binding protein increased the expression, in response to serum, of reporters under the control of the c-fos serum response element. Interestingly, the C-terminal moiety of the CREB-binding protein was not necessary to observe this effect. The cooperation did not require the Ets box in the serum response element, and the CArG box was sufficient, indicating that the CREB-binding protein is able to cooperate with p67(SRF) in the absence of an Ets protein. Co-immunoprecipitation experiments using cell extracts showed that p67(SRF) could be retained with antibodies directed against the CREB-binding protein, suggesting that the two proteins form a multimolecular complex in live cells. The physical interaction between p67(SRF) and the CREB-binding protein was further confirmed by two-hybrid assays in mammalian cells. Our results indicate that the CREB-binding protein cooperates with p67(SRF) and, thus, suggest that the serum response element is regulated by a multimolecular complex, which includes the CREB-binding protein, p67(SRF), and p62(TCF), with multiple interactions between the components of the complex.