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At least 1,549 records · Page 86Linked to original sources
PubMed2006

The role of activating protein 1 in the transcriptional regulation of the human FCGR2B promoter mediated by the -343 G -> C polymorphism associated with systemic lupus erythematosus.

The inhibitory receptor FcgammaRIIb is a negative regulator of antibody production and inflammatory responses. The -343 G --> C polymorphism in the human FCGR2B promoter is associated with systemic lupus erythematosus. The -343 C mutant promoter has decreased transcriptional activity. In the present study, we show that the transcriptional change correlates with quantitative differences in the interaction of the activating protein 1 complex with the mutant FCGR2B promoter. Promoter pulldown and chromatin immunoprecipitation assays demonstrated binding of c-Jun to the FCGR2B promoter. Phosphorylation of c-Jun was accompanied by transactivation of both FCGR2B promoter variants, whereas dephosphorylation of c-Jun by an inhibitor of c-Jun N-terminal kinase, markedly decreased the promoter activities. The -343 G --> C substitution enabled the specific interaction of the transcription factor Yin-Yang 1 with the mutant FCGR2B promoter. Yin-Yang 1 competed with activating protein 1 for binding at the -343 site, and contributed to the repression of the mutant FCGR2B promoter activity. This mechanism could be responsible for the decreased expression of FcgammaRIIb associated with the -343 C/C homozygous FCGR2B genotype in lupus patients. These findings provide a rationale for the transcriptional defect mediated by the -343 C/C FCGR2B promoter polymorphism associated with systemic lupus erythematosus, and add to our understanding of the complex transcriptional regulation of the human FCGR2B promoter.

Base Sequence↗
PubMed1991

Hybrid PLtl promoter with dual regulation control.

The newly constructed PLtl hybrid promoter is composed of the operator and promoter sequences of tac and the -35 to -135 region of the phage lambda PL promoter, which contains the AT-rich block and the OL2 and OL3 segments. Transcriptional properties of PLtl were examined and compared with tac and lambda PL as reference promoters. The hybrid PLtl exhibits different and improved properties over tac promoter in four ways: (i) when repressed, the repression is almost complete; (ii) after complete induction, the hybrid PLtl promoter shows a 1.4-2 times higher expression; (iii) the hybrid PLtl promoter permits flexible gene expression because it can be utilized under either or both repression controls simultaneously; (iv) the PLtl promoter permits enhanced expression of genes encoding products with unknown properties. When compared with the strong promoter PL from phage lambda, results with the PLtl promoter in lacZ fusion constructs show higher levels of beta-galactosidase activity. We also constructed a plasmid vector, pPLtl7G, which contains the hybrid PLtl promoter with a polylinker sequence at its 3' end, which facilitates efficient fusions of foreign genes in any of the reading frame.

Bacteriophage lambda↗
PubMed1995

Enhancer sharing in a plasmid model containing the alpha-fetoprotein and albumin promoters.

It has been proposed that the alpha-fetoprotein (AFP) enhancers also regulate the adjacent albumin gene, since the -10 kbp albumin enhancer is inactive in a number of cell lines that express albumin. In transfection experiments, the AFP enhancers strongly stimulate the albumin promoter in cells that silence the AFP promoter. These observations led us to develop a plasmid model of AFP-albumin gene switching, in which the albumin and AFP promoters would compete for the three AFP enhancers. However, when AFPCAT + ALBgal genes were combined with the AFP enhancers in one plasmid, both genes were driven at full activity. There was no change in the relative promoter expression over a wide range of transfected DNA concentrations, demonstrating that relative promoter activity was independent of DNA concentration and of promoter concentration, and that neither promoter was limiting the expression of the other. In contrast, a control plasmid containing two albumin promoters showed mutual inhibition, indicating the expected promoter competition. The albumin and AFP promoters noncompetitively shared the three enhancers on this plasmid, resulting in high levels of transcription from both promoters.

Albumins↗
PubMed1997

Liver-enriched transcription factors, HNF-1, HNF-3, and C/EBP, are major contributors to the strong activity of the chicken CYP2H1 promoter in chick embryo hepatocytes.

Chicken CYP2H1 promoter constructs express strongly in chick embryo hepatocytes at a level comparable with that of Rous sarcoma viral promoter. We have identified the transcription factors responsible for the active CYP2H1 promoter. Binding sites for transcription factors were located within the first 160 bp of promoter sequence using promoter deletion experiments and DNase I footprint analysis. Sequence analysis revealed characteristic sites for the liver-enriched transcription factors of the HNF-1, HNF-3, and C/EBP families and for the ubiquitous factor, USF. Protein binding to these sites was established by gel mobility shift assays. Mutagenesis and transient transfection experiments demonstrated that these sites, in combination, were responsible for the strong promoter activity with a substantial contribution from HNF-1 and HNF-3. The promoter was also active in mammalian HepG2 and COS-1 cell lines where expression was dependent on the identified transcription factor binding sites but promoter activity in the HeLa cells was low. Transactivation experiments revealed that promoter expression could be activated through the appropriate binding sites by exogenously expressed rat HNF-1alpha or HNF-1beta, rat HNF-3alpha or HNF-3beta and chicken C/EBP alpha. Transcriptional synergism between HNF-1 and C/EBP was observed in these transactivation experiments. A Barbie box-like sequence overlapped the USF element but was not functional. The results demonstrate that liver-enriched transcription factors and USF direct strong expression of the CYP2H1 promoter in transiently transfected cells. By comparison, in vivo expression of this gene in uninduced chick embryo hepatocytes is low but markedly increased by phenobarbital. Drug induction may therefore substantially reflect derepression of this inherently active promoter.

Animals↗
PubMed2005

Organization and promoter analysis of the zebrafish (Danio rerio) interferon gene.

Interferon plays important roles in confronting viral infections as the first line of defense. For the purpose of understanding the molecular mechanism which controls transcription of the interferon gene, we cloned and sequenced the interferon promoter region of the zebrafish interferon gene and characterized its activity using firefly luciferase transient transfection expression assays. Different fragments of the zebrafish interferon 5'-flanking region were transfected into ZFL cells. In these cell lines, maximum promoter activity was located in 2.2 kb of the zebrafish interferon 5' flanking region of the ZFL cell line. In this study, we investigated whether the viral replicative intermediate double-stranded RNA (herein we used synthetic polyinosinic-polycytidylic acid [poly(I):poly(C)] modifies the effects of interferon on gene expression. For this purpose, all zebrafish interferon promoter fragments were treated with either 1, 10, or 100 microg/ml poly(I):poly(C). The results showed that after treatment with 10 microg/ml poly(I):poly(C), high promoter activity was observed in the -2.2-kb interferon promoter fragment. Several putative transcription factors were shown in the promoter region, including IRF-1, C/EBP, NFkappaB, and GATA-1. Further study of the in vivo expression of the interferon promoter during development was carried out in transgenic zebrafish expressing an interferon promoter-driven green fluorescent protein (GFP) encoding the GFP cDNA transgene. Morphological studies of transgenic zebrafish indicated that the interferon promoter-driven GFP transcripts appeared in the yolk, head, and lymphoid organs. These results indicate that the interferon promoter is active in a tissue-specific manner, and suggest that the interferon promoter plays an important role in virus resistance during teleost growth.

Amino Acid Sequence↗
PubMed1996

Retroviral vector sequences may interact with some internal promoters and influence expression.

Although retroviral vectors show promise for gene therapy, their expression in animals has been low. An improved understanding of how promoters function from a retroviral vector should facilitate the design of improved vectors. In this study, liver-specific promoters were cloned into a retroviral vector and expression from the retroviral long terminal repeat (LTR) and the internal promoter was analyzed. In addition, oligomerized liver-specific transcription factor binding sites were placed upstream of each promoter in an attempt to increase expression further. Additional oligomerized binding sites only increased expression slightly or inhibited expression in hepatoma cells, suggesting that this is not an effective way to increase expression from a retroviral vector. Unexpectedly, the liver-specific albumin promoter was expressed at high levels from a retroviral vector in fibroblasts, suggesting that retroviral elements functioned as an enhancer. Furthermore, the addition of HNF-4 binding sites adjacent to the albumin promoter inhibited both the LTR and albumin promoter in fibroblasts, an effect that was probably mediated by inhibitory proteins present in nonhepatic cells that can bind to HNF-4 sites. These results suggest that both positive and negative influences can be transmitted between the LTR and the albumin promoter. In contrast, the liver-specific human alpha 1-antitrypsin promoter did not appear to interact with the LTR by either of these criteria. Retroviral vectors have sequences that may inhibit expression of the LTR and some internal promoters in vivo. We hypothesize that internal promoters that do not interact with the LTR in tissue culture will be resistant to inhibitory effects of retroviral sequences in vivo.

Albumins↗
PubMed1997

Position-dependent and promoter-specific regulation of gene expression in Trypanosoma brucei.

Trypanosoma brucei evades the mammalian immune response by a process of antigenic variation. This involves mutually exclusive and alternating expression of telomere-proximal variant surface glycoprotein genes (vsgs), which is controlled at the level of transcription. To examine transcription repression in T.brucei we inserted reporter genes, under the control of either rRNA or vsg expression site (ES) promoters, into various chromosomal loci. Position-dependent repression of both promoters was observed in the mammalian stage of the life cycle (bloodstream forms). Repression of promoters inserted into a silent ES was more pronounced closer to the telomere and was bi-directional. Transcription from both ES and rRNA promoters was also efficiently repressed at a non-telomeric vsg locus in bloodstream-form trypanosomes. In cultured tsetse fly midgut-stage (procyclic) trypanosomes, in which vsg is not normally expressed, all inserted rRNA promoters were derepressed but ES promoters remained silent. Our results suggest that vsg promoters and ectopic rRNA promoters in bloodstream-form T.brucei are restrained by position effects related to their proximity to vsgs or other features of the ES. Sequences present in rRNA promoters but absent from vsg ES promoters appear to be responsible for rRNA promoter-specific derepression in procyclic cells.

Animals↗
PubMed2003

The promoter of the heterochromatic Drosophila telomeric retrotransposon, HeT-A, is active when moved into euchromatic locations.

The Drosophila telomeric retrotransposon, HeT-A, is found only in heterochromatin; therefore, its promoter must function in this chromatin environment. Studies of position effect variegation suggest that promoters of heterochromatic genes are very different from euchromatic promoters, but this idea has not been tested with isolated promoter sequences. The HeT-A promoter is the first heterochromatin promoter to be isolated and it is of interest to investigate its activity when removed from telomeric heterochromatin. This promoter was initially characterized by testing reporter constructs in transient transfection of cultured cells, an environment that may approximate its endogenous heterochromatin. We now report P-element-mediated transpositions of these constructs, testing the function of different parts of the putative promoter in euchromatin. Expression of endogenous HeT-A RNA shows marked developmental regulation and accumulates preferentially in replicating diploid tissues. HeT-A promoter constructs are active in all euchromatic locations tested and some display aspects of endogenous HeT-A stage- and cell-type expression programs. The activity of each promoter construct in euchromatic locations is also generally consistent with its activity in the transient transfection tests; a possibly significant exception is one sequence segment that appreciably enhanced activity in transient transfection but repressed promoter activity in euchromatin.

ATP-Binding Cassette Transporters↗
PubMed2004

A study of safety-promoting activities for children and adolescents in 25 Swedish municipalities.

The purpose of this study was to investigate two elements of the community approach to safety promotion for children and adolescents. The first concerns whether there is any correlation between safety-promoting activities in different municipal settings, and the second whether socio-economic and demographic characteristics affect the level of safety-promoting activities. A cross-sectional study was performed comprising 25 municipalities in Stockholm County. The extent of safety-promoting activities was studied in four settings: the municipal setting in general, the traffic setting, the pre-school setting, and the school and leisure activity setting. Data on nine demographic and socio-economic characteristics of the municipalities were collected from public statistics. There were three main findings. First, safety-promoting activities in the municipal setting in general, the pre-school setting, and the school and leisure activity setting, but not in the traffic setting, were apparently associated with total municipal safety-promoting activities. Secondly, socio-economic characteristics, measured as the proportion of adults with >12 years of education, did not predict the extent of safety-promoting activities. Finally, growing municipalities with a large proportion of children reported fewer safety-promoting activities than non-growing municipalities. This study demonstrated weak correlations between safety-promoting activities in different municipal settings. The finding that growing municipalities have fewer safety-promoting activities provides pertinent lessons for future action and research. The study offers a method by which it may be possible to better assess the complex components of the community approach to safety promotion.

Adolescent↗
PubMed2005

A dinucleotide deletion in the ankyrin promoter alters gene expression, transcription initiation and TFIID complex formation in hereditary spherocytosis.

Ankyrin defects are the most common cause of hereditary spherocytosis (HS). In some HS patients, mutations in the ankyrin promoter have been hypothesized to lead to decreased ankyrin mRNA synthesis. The ankyrin erythroid promoter is a member of the most common class of mammalian promoters which lack conserved TATA, initiator or other promoter cis elements and have high G+C content, functional Sp1 binding sites and multiple transcription initiation sites. We identified a novel ankyrin gene promoter mutation, a TG deletion adjacent to a transcription initiation site, in a patient with ankyrin-linked HS and analyzed its effects on ankyrin expression. In vitro, the mutant promoter directed decreased levels of gene expression, altered transcription initiation site utilization and exhibited defective binding of TATA-binding protein (TBP) and TFIID complex formation. In a transgenic mouse model, the mutant ankyrin promoter led to abnormalities in gene expression, including decreased expression of a reporter gene and altered transcription initiation site utilization. These data indicate that the mutation alters ankyrin gene transcription and contributes to the HS phenotype by decreasing ankyrin gene synthesis via disruption of TFIID complex interactions with the ankyrin core promoter. These studies support the model that in promoters that lack conserved cis elements, the TFIID complex directs preinitiation complex formation at specific sites in core promoter DNA and provide the first evidence that disruption of TBP binding and TFIID complex formation in this type of promoter leads to alterations in start site utilization, decreased gene expression and a disease phenotype in vivo.

Ankyrins↗
PubMed2004

Activation of the Oryza sativa non-symbiotic haemoglobin-2 promoter by the cytokinin-regulated transcription factor, ARR1.

Using in silico methods, several putative phytohormone-responsive cis-elements in the Oryza sativa non-symbiotic haemoglobin (NSHB) 1-4 and Arabidopsis thaliana NSHB1-2 promoters have been identified. An OsNSHB2 promoter::GUS reporter gene fusion shows tissue-specific expression in A. thaliana. GUS expression was observed in roots, the vasculature of young leaves, in flowers, and in the pedicel/stem junction. In transient assays, activity of the OsNSHB2 promoter was significantly up-regulated in the presence of the cytokinin, 6-benzylaminopurine (BA). Deletion analyses indicated that the full-length promoter was required for maximal trans-activation in the presence of cytokinin. Mutation of the single cytokinin-regulated ARR1-binding element abolished promoter activation in response to cytokinin. Constitutive expression of ARR1 under the control of the 35S cauliflower mosaic virus promoter enhanced wild-type OsNSHB2 promoter activity, but had no effect on the activity of the mutated promoter in the absence of cytokinin. However, overexpression of ARR1 in the presence of cytokinin resulted in super-activation of the wild-type promoter. The mutated promoter was only moderately activated in the presence of cytokinin and ARR1, indicating that the OsNSHB2 promoter can be regulated by the ARR1 protein, but requires other cytokinin-induced factors for optimal activation. This is the first report that identifies a trans-acting factor involved in the activation of a NSHB gene.

Arabidopsis↗
PubMed2007

Chimpanzee, orangutan, mouse, and human cell cycle promoters exempt CCAAT boxes and CHR elements from interspecies differences.

Mechanisms regulating the cell division cycle are well conserved among all eukaryotes. Consistently many proteins regulating the cell cycle are functionally interchangeable between many organisms. Cell division control is regulated on different levels of which the transcriptional level appears to be particularly important for controlling synthesis of many cell cycle proteins. We had earlier described transcription factor-binding sites essential for regulating genes important for the transition from the G(2) phase to mitosis. A tandem repressor site named cell cycle-dependent element (CDE) and cell cycle genes homology region (CHR) are responsible for the correct expression during the cell cycle. Another feature of these G(2)/M-specific promoters is the activation through 2 or 3 CCAAT boxes binding the transcription factor nuclear factor-Y (NF-Y). These major activating sites have to be spaced 32 or 33 bp apart to be fully functional. We were interested in looking at the evolutionary changes in regulatory elements and overall promoter structure of 3 well-characterized cell cycle genes. Here, we compare the DNA sequences and functional features of the cdc25C, cyclin B1, and cyclin B2 promoters from humans, mouse, chimpanzee, and orangutan. We find numerous differences in the nucleotide sequence between mouse and primate promoters. However, CHR and CCAAT boxes stand out in that they are perfectly conserved in all promoters tested. The CDE site contains nucleotide exchanges between mouse and primate promoters. Comparing sequences and functions of chimpanzee, orangutan, and human promoters, we observe a complete conservation in nucleotide sequence of the regulatory elements. Functional assays of the cyclin B1, cyclin B2, and cdc25C promoters yield moderate variations in activity and thereby a good conservation of function. Although we find nucleotide differences in cell cycle promoters between orangutan and humans of about 5%, there are never changes in any of the CCAAT boxes or CDE/CHR sites in the cyclin B1, cyclin B2, and cdc25C promoters. Furthermore, we describe the influence of the tumor suppressor p53 and the transcriptional activator NF-Y on regulation of the newly cloned primate promoters.

Animals↗
PubMed1990

Study of Vitreoscilla globin (vgb) gene expression and promoter activity in E. coli through transcriptional fusion.

Bacterial hemoglobin (VtHb) is produced by the gram-negative bacterium, Vitreoscilla, in large quantity in response to hypoxic environmental conditions. The vgb gene coding for VtHb has been cloned in E. coli where it is expressed strongly by its natural promoter. The expression of the vgb gene in Vitreoscilla is transcriptionally regulated by oxygen. When E. coli cells were shifted from 20% to 5% oxygen, vgb specific transcript increased. In E. coli cells with plasmids carrying transcriptional fusions of the vgb gene promoter to either CAT (chloramphenicol acetyl transferase) or xylE (catechol-2,3-dioxygenase) genes, the promoter activity depended on the oxygen level. The concentration of CAT and xylE gene products in cells grown under 5% oxygen was 5-7 times that of aerobically (20% oxygen) grown cells. When the vgb gene promoter was deleted, VtHb was not produced under any conditions. When the promoter was replaced by the E. coli tac promoter, hypoxic oxygen did not affect the level of expression of vgb, but adding IPTG did increase the expression of this gene. These results indicate that the vgb gene promoter is transcriptionally regulated by oxygen even in E. coli, and that microaerobiosis is sufficient to induce vgb expression. The size of S1 nuclease-resistant hybrids, prepared using RNA transcripts protected with restriction enzyme fragments containing the promoter proximal region of vgb, was the same for both Vitreoscilla and E. coli, further evidence that the same promoter is used in both organisms. Transcriptional fusion of the vgb gene promoter to the xylE reporter gene on the broad host range plasmid, pKD-49, was used to demonstrate that the vgb promoter can be expressed in other gram-negative organisms, including Pseudomonas, Azotobacter, and Rhizobium.

Cloning, Molecular↗
PubMed1991

Protein-DNA interactions in the cAMP responsive promoter region of the murine ornithine decarboxylase gene.

To evaluate the function of the murine ornithine decarboxylase (ODC) gene promoter, expression of chimeric ODC-chloramphenicol acetyltransferase (CAT) plasmids (pODCcat) containing 1,658 nt of the ODC promoter sequence and its various 5'-deletions was analyzed. In transient expression assays with NIH/3T3 mouse cells, pODCcat constructs exhibited fairly strong promoter activity yielding CAT values up to 40% of those obtained with the viral promoter RSV. Interestingly, 5'-deletions of the pODCcat constructs increased the promoter activity over that achieved using the entire 1.6-kb 5'-flanking region, with the highest activity being observed with about 750 nt of the ODC promoter. This finding suggests that the distal part of the promoter includes DNA elements which are involved in repressing its function. The promoter region could be deleted down to the proximal 97 nt and still be stimulated by cAMP to the same extent as the 1.6-kb promoter. DNase I footprinting and methylation interference studies showed that a specific protein binds to the region from -59 to -39, which encompasses a DNA motif resembling the consensus cyclic AMP response element (CRE). However, comparative gel retardation and Southwestern blotting experiments with the putative ODC-CRE and the somatostatin promoter CRE indicated that the 70-kDa protein interacting with the CRE-like element of the ODC promoter is different from the well-characterized nuclear CRE-binding protein CREB.

Animals↗
PubMed1992

The yeast actin intron contains a cryptic promoter that can be switched on by preventing transcriptional interference.

We show that the single intron of the actin gene of the yeast Saccharomyces cerevisiae contains a cryptic promoter for transcription of the second exon. This promoter is inactive in the normal actin gene, but can be activated when the actin gene promoter is deleted. An identical activation was induced by placing efficient transcriptional terminators at position 61 of the 309 bp intron. In all cases transcripts with identical 5' ends close to the boundary of the intron and the second exon were produced. These results indicate that the cryptic promoter in the actin intron is occluded in the normal actin gene by transcriptional interference with the actin gene promoter. Transcription initiation near the intron/exon 2 boundary is enabled by protection from traversing polymerases, that initiated transcription at the upstream located actin gene promoter. A partial promoter protection using leaky terminators resulted in small amounts of transcripts initiated from the cryptic promoter. Although we do not know any function of the cryptic promoter in actin gene expression, it is tentative to speculate that the cryptic intron promoter might be a relict of a promoter that was functional earlier in evolution.

Actins↗
PubMed1993

Activation of a dual adenovirus promoter containing nonconsensus TATA motifs in Schizosaccharomyces pombe: role of TATA sequences in the efficiency of transcription.

The role of TATA elements in the expression of a mammalian promoter was investigated in the fission yeast Schizosaccharomyces pombe, by studying the human adenovirus E2-early promoter. This is a unique dual promoter with two nonconsensus TATA elements directing transcription from two cap sites, +1 and -26. A sequence TTAAGA provides the TATA box function for the +1 promoter, whereas a sequence TAAATT, with a closer resemblance to the consensus (TATAA/TA) provides this function for the -26 promoter. Yet, in human cells, the +1 promoter is transcribed about 20 fold more efficiently than the -26 promoter. We found that both promoters are transcribed faithfully in S. pombe with start sites identical or close to those found in human cells. Surprisingly, the relative ratio of expression for the +1 and -26 promoters was exactly reversed in S. pombe cells. This reversal appeared to be due to the relatively weak binding of S. pombe TATA binding protein to the TTAAGA motif, rather than to its rate of dissociation. Furthermore, we show that in S. pombe, promoter expression correlates well with the nucleotide sequence of the TATA element rather than the context in which it is placed. By contrast, it is the context of the TATA element, rather than its nucleotide sequence that appears to be critical for promoter expression in human cells. Our data suggest the existence of one or more additional factors in human cells that permit the utilization of nonconsensus TATA elements. S. pombe appears to lack these factors.

Adenoviruses, Human↗
PubMed1993

E. coli RNA polymerase, deleted in the C-terminal part of its alpha-subunit, interacts differently with the cAMP-CRP complex at the lacP1 and at the galP1 promoter.

A deletion of the C-terminal part of the alpha-subunit of RNA polymerase is known to affect differently promoters activated by CRP depending on the location of the CRP binding site at the promoter. When the CRP binding site is located at -61.5, as at lacP1 (a type I promoter), activation is strongly impaired while it is not significantly affected at galP1 where CRP binds 41.5 bp upstream of the start of the message (type II promoter). We have investigated the differences in the architecture of the corresponding open complexes by comparing the positioning of holoenzymes reconstituted respectively with native or with truncated alpha-subunits (containing the first 235 or 256 residues of a) at two 'up' promoter mutants of the lacP1 and galP1 promoters (respectively lacUV5 and gal9A16C). First, the affinity of wild-type RNA polymerase for both promoters is increased by the presence of CRP and cAMP. By contrast, holoenzymes reconstituted with truncated alpha-subunits, show cooperative binding at the galP1 promoter only. Second, footprinting data confirm these observations and indicate that the truncated holoenzymes are unable to recognize regions of the promoter upstream from position -40. The absence of contacts between the truncated enzymes and CRP at the lacP1 promoter can explain the deficiency in activation. At the galP1 promoter, where the CRP site is closer to the initiation site, protein-protein contacts can still occur with the truncated polymerases, showing that the C-terminal part of the alpha-subunit is not involved in activation.

Carrier Proteins↗
PubMed1994

The topology of the promoter of RNA polymerase II- and III-transcribed genes is modified by the methylation of 5'-CG-3' dinucleotides.

In eukaryotic cells, RNA polymerase II- and III-transcribed promoters can be inactivated by sequence-specific methylation. For some promoter motifs, the introduction of 5-methyldeoxycytidine (5-mC) residues has been shown to alter specific promoter motif-protein interactions. To what extent does the presence of 5-mC in promoter or regulatory DNA sequences affect the structure of DNA itself. We have investigated changes in DNA bending in three naturally occurring DNA elements, the late E2A promoter of adenovirus type 2 (Ad2) DNA, one of our main model systems, the VAI (virus-associated) RNA gene of Ad2 DNA, and an Alu element associated with the human angiogenin gene. Alterations in electrophoretic mobility of differently permuted promoter segments in non-denaturing polyacrylamide gels have been used as assay system. In the late E2A promoter of Ad2 DNA, a major and possibly some minor DNA bending motifs exist which cause deviations in electrophoretic mobility in comparison to coelectrophoresed marker DNA fragments devoid of DNA bending motifs. DNA elements have been specifically in vitro methylated by the HpaII (5'-CCGG-3'), the FnuDII (5'-CGCG-3'), or the CpG DNA methyltransferase from Spiroplasma species (M-SssI; 5'-CG-3'). Methylation by one of these DNA methyltransferases influences the electrophoretic mobility of the three tested promoter elements very strikingly, though to different extents. It cannot be predicted whether sequence-specific promoter methylation increases or decreases electrophoretic mobility; these changes have to be experimentally determined. Methylation of the E. coli dcm (5'-CCA/TGG-3') sites in some of the DNA constructs does not make a contribution to mobility changes. It is concluded that sequence-specific methylations in promoter or regulatory DNA elements can alter the bending of DNA very markedly. This parameter may contribute significantly to the silencing of promoters, probably via altering spatial relationships among DNA-bound transcription factors.

Adenovirus E2 Proteins↗