Space For Promotion Research Articles

Epigenetic regulation of TTF-I-mediated promoter–terminator interactions of rRNA genes

Ribosomal RNA synthesis is the eukaryotic cell's main transcriptional activity, but little is known about the chromatin domain organization and epigenetics of actively transcribed rRNA genes. Here, we show epigenetic and spatial organization of mouse rRNA genes at the molecular level. TTF-I-binding sites subdivide the rRNA transcription unit into functional chromatin domains and sharply delimit transcription factor occupancy. H2A.Z-containing nucleosomes occupy the spacer promoter next to a newly characterized TTF-I-binding site. The spacer and the promoter proximal TTF-I-binding sites demarcate the enhancer. DNA from both the enhancer and the coding region is hypomethylated in actively transcribed repeats. 3C analysis revealed an interaction between promoter and terminator regions, which brings the beginning and end of active rRNA genes into close contact. Reporter assays show that TTF-I mediates this interaction, thereby linking topology and epigenetic regulation of the rRNA genes.

Mapping of RNA Polymerase Residues that Interact with Bacteriophage Xp10 Transcription Antitermination Factor p7

Bacteriophage Xp10-encoded transcription factor p7 interacts with host Xanthomonas oryzae RNA polymerase β′ subunit and prevents both promoter recognition by the RNA polymerase holoenzyme and transcription termination by the RNA polymerase core. P7 does not bind to and has no effect on RNA polymerase from Escherichia coli. Here, we use a combination of biochemical and genetic methods to map the p7 interaction site to within four β′ amino acid residues at the N terminus of X. oryzae RNAP β′. The interaction site is located in an area that is close to the promoter spacer in the open complex and to the upstream boundary of the transcription bubble in the elongation complex, providing a possible explanation of how a small protein can affect both transcription initiation and termination by binding to the same RNA polymerase site.

Flagellar and global gene regulation in Helicobacter pylori modulated by changes in DNA supercoiling

In Helicobacter pylori, a host-adapted bacterium with a small genome and few dedicated transcriptional regulators, promoter structure, and gene organization suggested a role for DNA topology in the transcriptional regulation of flagellar genes. H. pylori DNA supercoiling, monitored by a reporter plasmid, was relaxed by novobiocin, an inhibitor of DNA gyrase. A decrease in negative supercoiling coincided with lowered transcription of the late flagellin gene flaA. Targeted mutagenesis that either increased or decreased promoter spacer length in the flaA σ 28 promoter lowered flaA transcript levels, expression of FlaA protein, and flagella formation. It also changed the promoter response to decreased superhelicity. Supercoiling of reporter plasmid DNA in H. pylori varied with growth phase in liquid culture. H. pylori σ 28 promoters of various spacer length, as well as other supercoiling-sensitive genes, were differentially transcribed during the growth phases, consistent with supercoiling being associated with growth phase regulation. Genome-wide transcript analysis of wild-type H. pylori under conditions of reduced supercoiling identified flagellar, housekeeping, and virulence genes, the expression of which correlated with supercoiling change and/or growth phase. These data indicate that global supercoiling changes may help coordinate temporal (growth phase-related) regulation of flagellar biosynthesis and other cellular functions in Helicobacter.

Comparative genomics of regulation of heavy metal resistance in Eubacteria

BackgroundHeavy metal resistance (HMR) in Eubacteria is regulated by a variety of systems including transcription factors from the MerR family (COG0789). The HMR systems are characterized by the complex signal structure (strong palindrome within a 19 or 20 bp promoter spacer), and usually consist of transporter and regulator genes. Some HMR regulons also include detoxification systems. The number of sequenced bacterial genomes is constantly increasing and even though HMR resistance regulons of the COG0789 type usually consist of few genes per genome, the computational analysis may contribute to the understanding of the cellular systems of metal detoxification.ResultsWe studied the mercury (MerR), copper (CueR and HmrR), cadmium (CadR), lead (PbrR), and zinc (ZntR) resistance systems and demonstrated that combining protein sequence analysis and analysis of DNA regulatory signals it was possible to distinguish metal-dependent members of COG0789, assign specificity towards particular metals to uncharacterized loci, and find new genes involved in the metal resistance, in particular, multicopper oxidase and copper chaperones, candidate cytochromes from the copper regulon, new cadmium transporters and, possibly, glutathione-S-transferases.ConclusionOur data indicate that the specificity of the COG0789 systems can be determined combining phylogenetic analysis and identification of DNA regulatory sites. Taking into account signal structure, we can adequately identify genes that are activated using the DNA bending-unbending mechanism. In the case of regulon members that do not reside in single loci, analysis of potential regulatory sites could be crucial for the correct annotation and prediction of the specificity.

Intergenic Transcripts Regulate the Epigenetic State of rRNA Genes

Transcripts originating from the intergenic spacer (IGS) that separates rRNA genes (rDNA) have been known for two decades; their biological role, however, is largely unknown. Here we show that IGS transcripts are required for establishing and maintaining a specific heterochromatic configuration at the promoter of a subset of rDNA arrays. The mechanism of action appears to be mediated through the interaction of TIP5, the large subunit of the chromatin remodeling complex NoRC, with 150-300 nucleotide RNAs that are complementary in sequence to the rDNA promoter. Mutations that abrogate RNA binding of TIP5 impair the association of NoRC with rDNA and fail to promote H3K9&H4K20 methylation and HP1 recruitment. Knockdown of IGS transcripts abolishes the nucleolar localization of NoRC, decreases DNA methylation, and enhances rDNA transcription. The results reveal an important contribution of processed IGS transcripts in chromatin structure and epigenetic control of the rDNA locus.

The porA pseudogene of Neisseria gonorrhoeae– low level of genetic polymorphism and a few, mainly identical, inactivating mutations

N. meningitidis is the only Neisseria species known to express two outer membrane porins, PorA and PorB. However, a porA pseudogene has been identified in N. gonorrhoeae. The present study investigated the prevalence and genetic polymorphism of this porA pseudogene in 87 different N. gonorrhoeae strains. The porA pseudogene was identified in all isolates. The pseudogene comprised 12 (5.5%), of which 10 were located in the promoter spacer, and 11 (1.0%) polymorphic nucleotide sites in the upstream segment containing the promoter region, i.e. the putative -10 and -35 sequences and the promoter spacer in-between, and the hypothetical PorA coding sequence, respectively. A phylogenetic analysis of the upstream segment and the hypothetical coding sequence identified 36 sequence variants, of which 30 were not previously described. All strains comprised at least two identical confirmed inactivating deletions, of which one was located in the promoter region and one in the hypothetical PorA coding sequence. In conclusion, the porA pseudogene and its few inactivating mutations are widespread in the N. gonorrhoeae population and the homology with the N. meningitidis porA gene reflects their common evolutionary origin. The highly conserved N. gonorrhoeae porA pseudogene may reflect an evolutionary neutral molecular clock and may be a suitable genetic target for diagnosis of N. gonorrhoeae.

Allele-specific germ cell epimutation in the spacer promoter of the 45S ribosomal RNA gene after Cr(III) exposure

Paternal exposure of mice to Cr(III) causes increased tumor risk in offspring; an epigenetic mechanism has been hypothesized. Representational difference analysis of gene methylation in sperm revealed hypomethylation in the 45S ribosomal RNA ( rRNA) gene after Cr(III) exposure, compared with controls. The most striking effects were seen in the rRNA spacer promoter, a region in the intergenic region of rRNA gene clusters that can influence transcription. Methylation of the rRNA spacer promoter has not been studied heretofore. Sperm DNAs from Cr(III)-treated and control mice were modified by the bisulfite method followed by PCR amplification of the spacer promoter, including 27 CpG sites. Cloning and dideoxy sequencing identified sequence variants (T or G at base −2214) in the spacer promoter. The T allele had less DNA methylation than the G allele in control mice (17 of 17 clones vs. 42 of 72 clones, P = 0.0004). In spite of diversity of sperm DNA methylation patterns, the DNA clones from Cr(III)-exposed mice had fewer methylated CpG sites, by an average of 19% ( P < 0.0001). This difference was limited to the G allele. The pyrosequencing technique was applied to quantify the percentage of methylation directly from amplified PCR products. Strikingly, for nine CpG sites including the spacer promoter core region, hypomethylation was highly significant in the Cr(III)-treated group (paired T test, P < 0.0001). Thus, one allele of the 45S rRNA spacer promoter is hypomethylated in sperm germ cells after Cr(III) exposure. This epimutation may lead to increase of tumor risk in the offspring.

Effects of distortions by A-tracts of promoter B-DNA spacer region on the kinetics of open complex formation by Escherichia coli RNA polymerase.

A-tracts in DNA due to their structural morphology distinctly different from the canonical B-DNA form play an important role in specific recognition of bacterial upstream promoter elements by the carboxyl terminal domain of RNA polymerase alpha subunit and, in turn, in the process of transcription initiation. They are only rarely found in the spacer promoter regions separating the -35 and -10 recognition hexamers. At present, the nature of the protein-DNA contacts formed between RNA polymerase and promoter DNA in transcription initiation can only be inferred from low resolution structural data and mutational and crosslinking experiments. To probe these contacts further, we constructed derivatives of a model Pa promoter bearing in the spacer region one or two An (n = 5 or 6) tracts, in phase with the DNA helical repeat, and studied the effects of thereby induced perturbation of promoter DNA structure on the kinetics of open complex (RPo) formation in vitro by Escherichia coli RNA polymerase. We found that the overall second-order rate constant ka of RPo formation, relative to that at the control promoter, was strongly reduced by one to two orders of magnitude only when the A-tracts were located in the nontemplate strand. A particularly strong 30-fold down effect on ka was exerted by nontemplate A-tracts in the -10 extended promoter region, where an involvement of nontemplate TG (-14, -15) sequence in a specific interaction with region 3 of sigma-subunit is postulated. A-tracts in the latter location caused also 3-fold slower isomerization of the first closed transcription complex into the intermediate one that precedes formation of RPo, and led to two-fold faster dissociation of the latter. All these findings are discussed in relation to recent structural and kinetic models of RPo formation.

Effects of distortions by A-tracts of promoter B-DNA spacer region on the kinetics of open complex formation by Escherichia coli RNA polymerase.

A-tracts in DNA due to their structural morphology distinctly different from the canonical B-DNA form play an important role in specific recognition of bacterial upstream promoter elements by the carboxyl terminal domain of RNA polymerase alpha subunit and, in turn, in the process of transcription initiation. They are only rarely found in the spacer promoter regions separating the -35 and -10 recognition hexamers. At present, the nature of the protein-DNA contacts formed between RNA polymerase and promoter DNA in transcription initiation can only be inferred from low resolution structural data and mutational and crosslinking experiments. To probe these contacts further, we constructed derivatives of a model Pa promoter bearing in the spacer region one or two An (n = 5 or 6) tracts, in phase with the DNA helical repeat, and studied the effects of thereby induced perturbation of promoter DNA structure on the kinetics of open complex (RPo) formation in vitro by Escherichia coli RNA polymerase. We found that the overall second-order rate constant ka of RPo formation, relative to that at the control promoter, was strongly reduced by one to two orders of magnitude only when the A-tracts were located in the nontemplate strand. A particularly strong 30-fold down effect on ka was exerted by nontemplate A-tracts in the -10 extended promoter region, where an involvement of nontemplate TG (-14, -15) sequence in a specific interaction with region 3 of sigma-subunit is postulated. A-tracts in the latter location caused also 3-fold slower isomerization of the first closed transcription complex into the intermediate one that precedes formation of RPo, and led to two-fold faster dissociation of the latter. All these findings are discussed in relation to recent structural and kinetic models of RPo formation.

Complete sequence of the 45-kb mouse ribosomal DNA repeat: analysis of the intergenic spacer☆

DNA from a single bacterial artificial chromosome clone was used to sequence the mouse ribosomal DNA intergenic spacer from the 3′ end of the 45S pre-RNA to the spacer promoter (Accession No. AF441733). This made possible the assembly of a complete mouse ribosomal DNA repeat unit (45,309 bp long, TPA Accession No. BK000964). Analysis of the intergenic spacer (IGS) showed a high density of simple sequence repeats and transposable elements. The IGS contains two long sequence blocks, which are repeated tandemly. Some of the sequences in these blocks are also present in other parts of the IGS. A difference in the mutation rate along the mouse IGS was observed. The significance of sequence motifs in the IGS for transcription enhancement, transcription termination, origin of replication, and nucleolar organization is discussed.

Comparison of PorA VR types and porA promoter sequence from Neisseria meningitidis B isolated from non-immunised children and vaccine failures immunised with a serogroup B outer membrane protein vaccine

PorA protein is an important component of group B meningococcal protein-based vaccines. The goals of this study were: (i) to classify the non-serosubtypable strains recovered from vaccine failures and controls by porA variable region (VR) type; (ii) to investigate if point mutations of VRs of the porA gene are present in P1.19,15 strains recovered from vaccine failures and controls; (iii) to investigate if nucleotide sequence variation in the promoter region of porA gene is related to low expression of PorA protein. VR type P1.19,15 predominated in younger vaccine failures (3–47 months) compared to older failures (48–83 months). No changes in VRs of porA were observed in 46 P1.19,15 strains studied. A promoter spacer of 16 bp and 10 guanidine residues in the polymeric G tract was detected in five of six strains with weak PorA expression. Overall, this study indicated that lack of antibody response was probably the major cause of low vaccine efficacy in young children.

Xenopus Ribosomal RNA Gene Intergenic Spacer Elements Conferring Transcriptional Enhancement and Nucleolar Dominance-like Competition in Oocytes

Repeated within the intergenic spacers that separate adjacent ribosomal RNA (rRNA) genes in Xenopus laevis are several distinct sequence elements. These include transcription terminators, "region 0" repeats, "region 1" repeats, duplicated spacer promoters, and 42-bp enhancer elements that are embedded within 60 or 81-bp repeats. All have been reported to stimulate RNA polymerase I transcription from an adjacent gene promoter. A greater number of 42-bp enhancers/gene have been suggested to explain the preferential transcription of X. laevis rRNA genes in X. laevis x Xenopus borealis hybrids, an epigenetic phenomenon known as nucleolar dominance. However, the possible contribution of regions 0/1 and/or spacer promoters to the preferential transcription of X. laevis (over X. borealis) rRNA genes has never been tested directly. In this study, we systematically tested the various intergenic spacer elements for their contributions to promoter strength and nucleolar dominance-like competition in oocytes. In disagreement with a previous report, region 0 and region 1 repeats do not have significant enhancer activity, nor do they play a discernible role in X. laevis-X. borealis rRNA gene competition. Minigenes containing X. laevis spacer sequences are only dominant over minigenes having complete X. borealis spacers if a spacer promoter is located upstream of the 42-bp enhancers; X. laevis enhancers alone are not sufficient. These results provide additional evidence that spacer promoters together with adjacent enhancers form a functional activating unit in Xenopus oocytes.

Variable surface protein Vmm of Mycoplasma mycoides subsp. mycoides small colony type.

A variable surface protein, Vmm, of the bovine pathogen Mycoplasma mycoides subsp. mycoides small colony type (M. mycoides SC) has been identified and characterized. Vmm was specific for the SC biotype and was expressed by 68 of 69 analyzed M. mycoides SC strains. The protein was found to undergo reversible phase variation at a frequency of 9 x 10(-4) to 5 x 10(-5) per cell per generation. The vmm gene was present in all of the 69 tested M. mycoides SC strains and encodes a lipoprotein precursor of 59 amino acids (aa), where the mature protein was predicted to be 36 aa and was anchored to the membrane by only the lipid moiety, as no transmembrane region could be identified. DNA sequencing of the vmm gene region from ON and OFF clones showed that the expression of Vmm was regulated at the transcriptional level by dinucleotide insertions or deletions in a repetitive region of the promoter spacer. Vmm-like genes were also found in four closely related mycoplasmas, Mycoplasma capricolum subsp. capricolum, M. capricolum subsp. capripneumoniae, Mycoplasma sp. bovine serogroup 7, and Mycoplasma putrefaciens. However, Vmm could not be detected in whole-cell lysates of these species, suggesting that the proteins encoded by the vmm-like genes lack the binding epitope for the monoclonal antibody used in this study or, alternatively, that the Vmm-like proteins were not expressed.

Monomer-dimer control of the ColE1 P(cer) promoter.

XerCD-mediated recombination at cer converts multimers of plasmid ColE1 to monomers, maximizing the number of independently segregating molecules and minimizing the frequency of plasmid loss. In addition to XerCD, recombination requires the accessory factors ArgR and PepA. The promoter P(cer), located centrally within cer, is also required for stable plasmid maintenance. P(cer) is active in plasmid multimers and directs transcription of a short RNA, Rcd, which appears to inhibit cell division. It has been proposed that Rcd is part of a checkpoint which ensures that multimer resolution is complete before the cell divides. This study has shown that ArgR does not act as a transcriptional repressor of P(cer) in plasmid monomers. P(cer) is unusual in that the -35 and -10 hexamers are separated by only 15 bp and this study has demonstrated that increasing this to a more conventional spacing results in elevated activity. An increase to 17 bp resulted in a 10- to 20-fold increase in activity, while smaller effects were seen when the spacer was increased to 16 bp or 18 bp. These observations are consistent with the hypothesis that P(cer) activation involves realignment of the -35 and -10 sequences within a recombinational synaptic complex. This predicts that a 17 bp spacer promoter derivative should be down-regulated by plasmid multimerization, and this is confirmed experimentally.

Multiple mechanisms of phase variation of PorA in Neisseria meningitidis.

Previously, we reported that PorA expression in Neisseria meningitidis is modulated by variation in the length of the homopolymeric tract of guanidine residues between the -35 and -10 regions of the promoter or by deletion of porA. To reveal additional mechanisms of variation in PorA expression, the meningococcal isolates from 41 patients and 19 carriers were studied. In addition, at least 3 meningococcal isolates from different body parts of each of 11 patients were analyzed. Sequence analysis of the porA promoter showed that the spacer between the -35 and -10 regions varies in length between 14 and 24 bp. PorA expression was observed in strains with a porA promoter spacer of 16 to 24 bp. All but one strain with a porA promoter spacer of 16 to 20 bp and undetectable PorA expression have a homopolymeric tract of 8 or 6 instead of 7 adenine residues in the porA coding region. The other PorA-negative strain had a single-base-pair deletion in the coding region. The highest level of PorA expression was observed in strains with a promoter spacer of 17 or 18 bp. PorA expression was reduced twofold in strains with a porA promoter spacer of 16 or 19 bp. Strains with a 16-bp promoter spacer with substitutions in the polyguanidine tract displayed increased levels of PorA expression compared to strains with a homopolymeric tract of guanidine residues in the porA promoter. In conclusion, meningococci display multiple mechanisms for varying PorA expression.

25S-18S rDNA intergenic spacer ofNicotiana sylvestris (Solanaceae): Primary and secondary structure analysis

The complete nucleotide sequence (3701 bp) of the intergenic spacer region between the 3′-end of 25S rRNA gene and the 5′-end of 18S rRNA gene (IGS) is described forNicotiana sylvestris. The IGS ofN. sylvestris consists of seven structural regions (SR) two of which are composed of subrepeats (SR II: three and one half copies of the 76 bp D-subrepeat and numerous short C-subrepeats; SR IV: five copies of the 142/136 bp A-subrepeat). A presumptive rRNA processing signal (CTTTT) is located at the beginning of SR I. An identical sequence is also present in each copy of the A-subrepeat. Each D-subrepeat in SR IIa contains at the 5′-end the conserved sequence (GAGGTTTTT) which may be involved in transcription termination. SR III (TG-rich) and SR IV (AT-rich) in the central part of IGS contain several potential replication origin sites. The transcription initiation site (TATATAAGGGGGG) is located at the border of SR IV and V. No spacer promoters have been found. SR V contains four copies of a CATGG motif that is also present in SR II, VI and VII. At the 5′-end of SR VII there are three copies of the 66 bp highly divergent B-subrepeat. Presumptive pre-rRNA processing signals are also present here. Possible hair-pin structures are described for SR V, VI and VII.

A ribosomal orphon sequence from Xenopus laevis flanked by novel low copy number repetitive elements.

We have used a differential cloning approach to isolate ribosomal/non-ribosomal frontier sequences from Xenopus laevis. A ribosomal intergenic spacer sequence (IGS) was cloned and shown not to be physically linked with the ribosomal locus. This ribosomal orphon contained the IGS sequences found immediately downstream of the 28S gene and included an array of enhancer repetitions and a non-functional spacer promoter. The orphon sequence was flanked by a member of the novel 'Frt' low copy repetitive element family. Three individual Frt repeats were sequenced and all members of this family were shown to lie clustered at two chromosomal sites, one of which contained the ribosomal orphon. One of the Frt elements contained an insertion of 297 bp that showed extensive homology to sequences within at least three other Xenopus genes. Each homology region was flanked by members of the T2 family of short interspersed repetitive elements, (SINEs), and by its target insertion sequence, suggesting multiple translocation events. The data are discussed in terms of the evolution of the ribosomal gene locus.

Spacing of promoter elements regulates the basal expression of the soxS gene and converts SoxR from a transcriptional activator into a repressor.

SoxR protein of Escherichia coli governs a global response against superoxide-generating agents (such as paraquat) or nitric oxide, and provides broad antibiotic resistance. A redox signal activates SoxR post-translationally to trigger transcription of a second regulatory gene, soxS. Activated and non-activated SoxR bind the soxS promoter with the same high affinity, but only the activated protein stimulates soxS transcription. SoxR acts by an unusual mechanism of positive control: the protein binds the soxS promoter between near-consensus -10 and -35 elements that are separated by an unusually long 19 bp (versus the optimal 17 bp). We have constructed and analyzed site-specific deletions that alter the promoter element spacing. Reducing the spacer length to 16-18 bp dramatically elevated basal expression of soxS in vivo and in vitro, and nearly eliminated additional activation by SoxR in response to paraquat. More strikingly, shortening the spacer converted SoxR from an activator into a repressor regardless of paraquat treatment. Gel mobility-shift assays show that repression by SoxR of the promoters with 17 and 16 bp spacers is due to interference with binding by RNA polymerase. Thus, activated SoxR remodels the unusual configuration of the wild-type soxS promoter into a highly active form, probably by compensating for the suboptimal distance between the -10 and the -35 elements.

Structural analysis of mouse rDNA: coincidence between nuclease hypersensitive sites, DNA curvature and regulatory elements in the intergenic spacer.

We have analyzed the chromatin structure of mouse ribosomal RNA genes (rDNA) by partial digestion of genomic DNA with micrococcal nuclease (MNase), DNase I and identified hypersensitive sites by indirect end-labeling. This analysis has revealed defined regions of nuclease hypersensitivity in the intergenic spacer which in turn coincide with regulatory elements. Hypersensitive sites map to the transcription initiation site, the enhancer repeats, the spacer promoter and two sequence elements which coincide with amplification-promoting sequences. Analysis of the DNA curvature by computer modeling uncovered a striking correlation between sequence-directed structural features of regulatory regions and the position of nuclease hypersensitive sites. Moreover, we demonstrate that nucleosomes are specifically positioned upstream and downstream of the transcription start site. In vitro studies using chromatin assembled in the presence of Drosophila embryo extracts show that binding of the transcription termination factor TTF-I to the upstream terminator mediates this specific nucleosome positioning at the rDNA promoter in an ATP- dependent fashion.

Virtually the Entire Xenopus laevis rDNA Multikilobase Intergenic Spacer Serves to Stimulate Polymerase I Transcription

The promoter-distal half of the spacer separating the tandem Xenopus laevis rRNA genes consists of "0" and "1" repetitive elements that have been considered unimportant in polymerase I transcriptional activation. Utilizing oocyte microinjection, we now demonstrate that the 0/1 region, as well as its component 0 and 1 repeats, substantially stimulate transcription from a ribosomal promoter in cis and inhibit transcription when located in trans. Both the cis and trans responses increase linearly with increasing numbers of 0 or 1 repeats until saturation is approached. The 0/1 block and its component elements stimulate transcription in both orientations, over distances, and when placed downstream of the initiation site, properties for which the 60/81-base pair (bp) repeats have been defined as polymerase I enhancers. In their natural promoter-distal rDNA location, the 0/1 repeats can stimulate transcription from the rRNA gene promoter, above the level afforded by the intervening 60/81-bp repeats and spacer promoter. In addition, as with the 60/81-bp repeats, the 0/1 repeats bind a factor in common with the rDNA promoter. Thus, the entire X. laevis rDNA intergenic spacer (the 0 repeats, 1 repeats, spacer promoter repeats, and 60/81-bp repeats) acts together to enhance ribosomal transcription.