Differences In Promoter Sequences Research Articles

The SIDT2/MDA5/IFN axis contributes to virus resistance in teleost fish

Disease resistance varies among fish species. The genetic factors contributing to disease resistance are largely unknown. Here, by comparative analysis on the disease resistance trait of grass carp (Ctenopharyngodon idellus, Ci) and barbel chub (Squaliobarbus curriculus, Sc), we report that the genes encoding SIDT2, MDA5 and IFN are associated with the resistance to viral infection. We identified the amino acid difference of SIDT2s, the existing or lacking of dsRNA binding loci in the carboxy-terminal domain (CTD) of MDA5s, and the promoter difference of ifn2s between the two fish species. Both CiSIDT2 and ScSIDT2 negatively regulate the MDA5-IRF3/7 mediated type I IFN reaction, and ScSIDT2 exhibited a stronger ability than CiSIDT2 to induce Ciirf7, Ciifn1, Ciifn2 and Cimx expressions, and to resist GCRV infection. ScMDA5 showed a greater ability than CiMDA5 in the induction of IRF7-mediated type I interferon and anti-GCRV function. CiMDA5 CTD inhibited Ciifn1 while enhanced Ciifn2 exrpessions, and ScMDA5 CTD exerts opposite effects on Ciifn1 and Ciifn2 expressions. ScMDA5 CTD possesses the ability to bind with GCRV nucleic acid fragments, while CiMDA5 lacking binding with virus genome. The promoter activities of Ciifn2 is significantly higher than that of Scifn2 owning to their differences in promoter sequences and binding proteins. These data revealed the mechanism of SIDT2-MDA5-IFN genetic architecture contributing to the difference in interspecies resistance and opening the possibility of molecular breeding for fish resistance.

Functional genomic analysis of K+ related salt-responsive transporters in tolerant and sensitive genotypes of rice.

Salinity is a complex environmental stress that affects the growth and production of rice worldwide. But there are some rice landraces in coastal regions that can survive in presence of highly saline conditions. An understanding of the molecular attributes contributing to the salinity tolerance of these genotypes is important for developing salt-tolerant high yielding modern genotypes to ensure food security. Therefore, we investigated the role and functional differences of two K+ salt-responsive transporters. These are OsTPKa or Vacuolar two-pore potassium channel and OsHAK_like or a hypothetical protein of the HAK family. These transporters were selected from previously identified QTLs from the tolerant rice landrace genotype (Horkuch) and sensitive genotype (IR29). In silico comparative sequence analysis of the promoter sequences of two these genes between Horkuch and IR29 was done. Real-Time expression of the selected genes in leaves and roots of IR29 (salt-sensitive), I-14 and I-71 (Recombinant Inbred Lines of IR29(♀)× Horkuch), Horkuch and Pokkali (salt-tolerant) under salt-stress at different time points was analyzed. For further insight, OsTPKa and OsHAK_like were chosen for loss-of-function genomic analysis in Horkuch using the CRISPR/Cas9 tool. Furthermore, OsTPKa was chosen for cloning into a sensitive variety by Gateway technology to observe the effect of gain-of-function. The promoter sequences of the OsTPKa and OsHAK_like genes showed some significant differences in promoter sequences which may give a survival advantage to Horkuch under salt-stress. These two genes were also found to be overexpressed in tolerant varieties (Horkuch and Pokkali). Moreover, a coordinated expression pattern between these two genes was observed in tolerant Horkuch under salt-stress. Independently transformed plants where the expression of these genes was significantly lowered, performed poorly in physiological tests for salinity tolerance. On the other hand, positively transformed T0 plants with the OsTPKa gene from Horkuch consistently showed growth advantage under both control and salt stress. The poor performance of the transgenic plants with the down-regulated genes OsTPKa and OsHAK_like under salt stress supports the assumption that OsTPKa and OsHAK_like play important roles in defending the rice landrace Horkuch against salt stress, minimizing salt injury, and maintaining plant growth. Moreover, the growth advantage provided by overexpression of the vacuolar OsTPKa K+ transporter, particularly under salt stress reconfirms its important role in providing salt tolerance. The QTL locus from Horkuch containing these two transporters maybe bred into commercial rice to produce high-yielding salt tolerant rice.

Deconvolution of intergenic polymorphisms determining high expression of Factor H binding protein in meningococcus and their association with invasive disease.

Neisseria meningitidis is a strictly human pathogen and is the major cause of septicemia and meningitis worldwide. Factor H binding protein (fHbp) is a meningococcal surface-exposed lipoprotein that binds the human Complement factor H allowing the bacterium to evade the host innate immune response. FHbp is also a key antigen in two vaccines against N. meningitidis serogroup B. Although the fHbp gene is present in most circulating meningococcal strains, level of fHbp expression varies among isolates and has been correlated to differences in promoter sequences upstream of the gene. Here we elucidated the sequence determinants that control fHbp expression in globally circulating strains. We analyzed the upstream fHbp intergenic region (fIR) of more than 5800 strains representative of the UK circulating isolates and we identified eleven fIR sequence alleles which represent 88% of meningococcal strains. By engineering isogenic recombinant strains where fHbp expression was under the control of each of the eleven fIR alleles, we confirmed that the fIR sequence determines a specific and distinct level of expression. Moreover, we identified the molecular basis for variation in expression through polymorphisms within key regulatory regions that are known to affect fHbp expression. We experimentally established three expression groups, high-medium-low, that correlated directly with the susceptibility to killing mediated by anti-fHbp antibodies and the ability of the meningococcal strain to survive within human serum. By using this sequence classification and information about the variant, we predicted fHbp expression in the panel of UK strains and we observed that strains with higher expressing fIR alleles are more likely associated with invasive disease. Overall, our findings can contribute to understand and predict vaccine coverage mediated by fHbp as well as to shed light on the role of this virulence factor in determining an invasive phenotype.

DNA structural features of eukaryotic TATA‐containing and TATA‐less promoters

Eukaryotic genes can be broadly classified as TATA‐containing and TATA‐less based on the presence of TATA box in their promoters. Experiments on both classes of genes have revealed a disparity in the regulation of gene expression and cellular functions between the two classes. In this study, we report characteristic differences in promoter sequences and associated structural properties of the two categories of genes in six different eukaryotes. We have analyzed three structural features, DNA duplex stability, bendability, and curvature along with the distribution of A‐tracts, G‐quadruplex motifs, and CpG islands. The structural feature analyses reveal that while the two classes of gene promoters are distinctly different from each other, the properties are also distinguishable across the six organisms.

WEIGHTED ALIGNMENT FREE DISSIMILARITY METRIC FOR PROMOTER SEQUENCE COMPARISON

Comparative sequence analysis has been a powerful tool in bioinformatics which interprets knowledge about the functionality of a sequence, making use of its structural information. Among the non coding regions of DNA, the comparison of promoter sequences has received a great deal of attention in medical science as promoter regions play a crucial role in gene regulation. In this work we propose an alignment free sequence comparison metric for comparison of promoter sequences. We use the binary and decimal position specific motif matrices (PSMM) of the promoters which were created for our experiments using the TFSEARCH tool. Simple weighted algorithm is used to compute the dissimilarity between the PSMMs of promoter sequences, thereby analyzing its underlying homology and functionality. The NCBI database was used to obtain the promoter sequences of 500 nucleotides upstream the transcription start site (TSS) of enzyme pyruvate kinase (PKLR) from the glycolysis pathway of different organisms for one experiment and all the enzymes from the glycolysis pathway of organism human for the other. The proposed dissimilarity metric is successful in bringing out differences on both the datasets and the results regarding similarities and differences in promoter sequences could be essential to have a clear knowledge of transcription regulation process in different organisms.The results reveal some useful findings which can be extended for a broader investigation.

Transcription start site sequence and spacing between the -10 region and the start site affect reiterative transcription-mediated regulation of gene expression in Escherichia coli.

Reiterative transcription is a reaction catalyzed by RNA polymerase, in which nucleotides are repetitively added to the 3' end of a nascent transcript due to upstream slippage of the transcript without movement of the DNA template. In Escherichia coli, the expression of several operons is regulated through mechanisms in which high intracellular levels of UTP promote reiterative transcription that adds extra U residues to the 3' end of a nascent transcript during transcription initiation. Immediately following the addition of one or more extra U residues, the nascent transcripts are released from the transcription initiation complex, thereby reducing the level of gene expression. Therefore, gene expression can be regulated by internal UTP levels, which reflect the availability of external pyrimidine sources. The magnitude of gene regulation by these mechanisms varies considerably, even when control mechanisms are analogous. These variations apparently are due to differences in promoter sequences. One of the operons regulated (in part) by UTP-sensitive reiterative transcription in E. coli is the carAB operon, which encodes the first enzyme in the pyrimidine nucleotide biosynthetic pathway. In this study, we used the carAB operon to examine the effects of nucleotide sequence at and near the transcription start site and spacing between the start site and -10 region of the promoter on reiterative transcription and gene regulation. Our results indicate that these variables are important determinants in establishing the extent of reiterative transcription, levels of productive transcription, and range of gene regulation.

Transcriptional Regulation of Two Conceptus Interferon Tau Genes Expressed in Japanese Black Cattle during Peri-Implantation Period

Interferon tau (IFNT), produced by the mononuclear trophectoderm, signals the process of maternal recognition of pregnancy in ruminants. However, its expression in vivo and its transcriptional regulation are not yet well characterized. Objectives of this study were to determine conceptus IFNT gene isoforms expressed in the bovine uterus and to identify differences in promoter sequences of IFNT genes that differ in their expression. RNA-seq data analysis of bovine conceptuses on days 17, 20, and 22 (day 0 = day of estrus) detected the expression of two IFNT transcripts, IFNT1 and IFNTc1, which were indeed classified into the IFNT gene clade. RNA-seq and quantitative RT-PCR analyses also revealed that the expression levels of both IFNT mRNAs were highest on day 17, and then decreased on days 20 and 22. Bovine ear-derived fibroblast (EF) cells, a model system commonly used for bovine IFNT gene transcription study in this laboratory, were cotransfected with luciferase reporter constructs carrying upstream (positions −637 to +51) regions of IFNT1 or IFNTc1 gene and various transcription factor expression plasmids including CDX2, AP-1 (Jun) and ETS2. CDX2, either alone or with the other transcription factors, markedly increased luciferase activity. The upstream regions of IFNT1 and IFNTc1 loci were then serially deleted or point-mutated at potential CDX-, AP-1-, and ETS-binding sites. Compared to the wild-type constructs, deletion or mutation at CDX2 or ETS2 binding sites similarly reduced the luciferase activities of IFNT1- or IFNTc1-promoter constructs. However, with the AP-1 site mutated construct, IFNT1- and IFNTc1-reporters behaved differently. These results suggest that two forms of bovine conceptus IFNT genes are expressed in utero and their transcriptional regulations differ.

DNA methylation mapping by tag-modified bisulfite genomic sequencing

A tag-modified bisulfite genomic sequencing (tBGS) method employing direct cycle sequencing of polymerase chain reaction (PCR) products at kilobase scale, without conventional DNA fragment cloning, was developed for simplified evaluation of DNA methylation sites. The method entails subjecting bisulfite-modified genomic DNA to a second-round PCR amplification employing GC-tagged primers. Qualitative results from tBGS closely correlated with those from conventional BGS ( R = 0.935, p = 0.002). In application, the intertissue and interindividual CpG methylation differences in promoter sequence for two genes, CYP1B1 and GSTP1, were then explored across four human tissue types (peripheral blood cells, exfoliated buccal cells, paired nontumor–tumor lung tissues), and two lung cell types in culture (normal NHBE and malignant A549). Predominantly conserved methylation maps for the two gene promoters were apparent across donors and tissues. At any given CpG site, variation in the degree of methylation could be determined by the relative height of C and T peaks in the sequencing trace. Methylation maps for the GSTP1 promoter diverged between NHBE (unmethylated) and A549 (completely methylated) cells in a previously unexplored upstream region, correlating with a 2.7-fold difference in GSTP1 mRNA expression ( p < 0.01). The tBGS method simplifies detailed methylation scanning of kilobase-scale genomic DNA, facilitating more ambitious genomic methylation mapping studies.

The role of vasopressin in the genetic and neural regulation of monogamy.

Arginine vasopressin modulates pairbond formation in the monogamous prairie vole (Microtus ochrogaster). Our laboratory has investigated the genetic and neural mechanisms by which vasopressin and its V1a receptor (V1aR) regulate social attachment between mates. Non-monogamous vole species show strikingly different distribution patterns of brain V1aR expression compared to monogamous species, and these patterns are thought to arise from species differences in the respective promoter sequences of the V1aR gene. Individual differences in prairie vole V1aR patterns may also reflect individual differences in promoter sequences. Pharmacological and genetic manipulation of the specific brain regions that express V1aR in the 'monogamous pattern' allows multilevel examination of the neural circuits that underlie pairbond formation in monogamous species. For example, V1aR are expressed in brain regions involved in reward circuitry in monogamous vole species and have been implicated in pairbonding. V1aR are also highly expressed in regions implicated in the olfactory processing of sociosexual behaviour. We hypothesize that both circuits of reward and olfactory memory underlie the cognitive mechanisms that control pairbonding. When used in conjuction, genetic and cellular analyses of a complex social behaviour can provide a coherent framework with which to examine the role of the vasopressin system in species evolution and neural control of behaviour.

Regulation of Staphylococcus aureus type 5 and type 8 capsular polysaccharides by CO(2).

Staphylococcus aureus expression of capsular polysaccharide type 5 (CP5) has been shown to be downregulated by CO(2). Here we show that CO(2) reduces CP5 expression at the transcriptional level and that CO(2) regulates CP8 expression depending on the genetic background of the strains. Growth in the presence of air supplemented with 5% CO(2) caused a significant decrease in CP8 expression in four S. aureus strains, a marginal effect in four strains, and higher CP8 expression in strain Becker. Absolute CP8 expression in the nine S. aureus strains differed largely from strain to strain. Four groups of strains were established due to sequence variations in the promoter region of cap5 and cap8. To test whether these sequence variations are responsible for the different responses to CO(2), promoter regions from selected strains were fused to the reporter gene xylE in pLC4, and the plasmids were electrotransformed into strains Becker and Newman. XylE activity was negatively regulated by CO(2) in all derivatives of strain Newman and was always positively regulated by CO(2) in all derivatives of strain Becker. Differences in promoter sequences did not influence the pattern of CP8 expression. Therefore, the genetic background of the strains rather than differences in the promoter sequence determines the CO(2) response. trans-acting regulatory molecules may be differentially expressed in strain Becker versus strain Newman. The strain dependency of the CP8 expression established in vitro was also seen in lung tissue sections of patients with cystic fibrosis infected with CP8-positive S. aureus strains.

Prolactin induction of insulin gene transcription: roles of glucose and signal transducer and activator of transcription 5.

GH and PRL stimulate insulin production in pancreatic beta-cells through induction of insulin gene transcription. The transcriptional effects of GH are mediated through the binding of signal transducer and activator of transcription-5 (STAT5) to a consensus recognition sequence (TTCnnnGAA) in the rat insulin-1 promoter. In this study we demonstrate that PRL also induces the binding of STAT5 proteins to the rat insulin-1 STAT5 motif. However, the magnitude of binding of STAT5 nuclear proteins, as assessed by electrophoretic mobility shift assays, was only 1/30th that of the binding of the same STAT5 proteins to the beta-casein STAT5 site. The differences in the affinities of the rat insulin-1 and beta-casein STAT5 motifs are explained in part by differences in promoter sequences flanking the STAT5 sites. To assess the importance of the STAT motif in PRL induction of insulin gene transcription, we deleted the STAT5 consensus sequence in the rat insulin 1 promoter, cloned the truncated promoter upstream of the luciferase reporter gene, and transfected the construct into rat insulinoma (INS-1) cells. The transcriptional activity of this construct was compared with that of the wild-type promoter. Although deletion of the STAT5 site in the promoter reduced the basal luciferase activity, the response to PRL was unaffected. PRL also induced transcription of constructs containing the wild-type human insulin promoter or the rat insulin-2 promoter, which contain no classic STAT5 sequences. The transcriptional effect of PRL was manifest even when cells were incubated in glucose-free medium, indicating that the action of the hormone is not mediated solely through changes in glucose uptake or glucose metabolism. To identify PRL-responsive regions of the rat and human insulin promoters, we constructed a series of promoter truncations and assessed their responsiveness to PRL. A PRL-responsive region of the rat insulin-1 promoter was localized between nucleotides -165 and -109. A PRL-responsive region of the human insulin promoter was localized between nucleotides -346 and -250. Additional regions of the human and rat insulin-1 promoters were required for PRL induction of a heterologous, minimal thymidine kinase promoter, suggesting that there are multiple PRL-responsive elements in the insulin genes. These observations suggest a glucose- and STAT5-independent pathway by which PRL may induce insulin gene transcription.

Differential Regulation of Vitronectin in Mice and Humans in Vitro

To define the cis-acting elements involved in the regulation of the murine vitronectin (Vn) gene in inflammation, the 5'-flanking region was isolated, fused to the luciferase reporter gene, and the basal and interleukin 6 (IL-6)-stimulated transcriptional activity was tested in transfection experiments using Hep3B cells. Treatment with IL-6 induced this construct by more than 20-fold, whereas the corresponding 5'-flanking region of the human Vn gene was not stimulated. Transfection studies using murine Vn constructs with serial 5'-deletions revealed that two sequences were important in the IL-6 response, and specific mutations in both sequences abolished the response. A 2-base pair mutation converted the human sequence to that of a murine IL-6 responsive element and partially conveyed IL-6 inducibility. In contrast, transforming growth factor beta stimulated the human construct and the endogenous Vn gene in human Hep3B cells in a dose-dependent manner, whereas the murine construct was not responsive. The transforming growth factor beta responsive region was localized to a 30-base pair fragment with little homology to the murine sequence. These studies reveal that the structural basis for the differential regulation of the human and murine Vn genes resides in the differences in promoter sequence.

The different level of expression of HLA-DRB1 and -DRB3 genes is controlled by conserved isotypic differences in promoter sequence

HLA-DRB1 and -DRB3 are two genes encoding two distinct HLA-DR β chains in the DRw52 family of haplotypes (DR3, DR5, DR13, and DR14). These β chains determine the structural and functional identity of the two kinds of HLA-DR molecules expressed. The highly polymorphic HLA-DRB1 locus is always expressed at a higher level than the HLA-DRB3 locus, and functional assays indicate that the proximal promoter of DRB1 is indeed more active than that of DRB3. The DNA sequence of the two promoters in nine different DRw52 haplotypes has revealed a stricking allelic conservation as well as characteristic, isotype-specific, conserved-sequence motifs. These isotype-specific differences concern the functionally essential X and Y box motifs of HLA class II promoters, and they do indeed affect binding of specific nuclear factors to the X and Y boxes of DRB1 or DRB3 promoters. Finally, analysis of the activity of various normal and mutated DRB1 or DRB3 promoters indicates that the X box region of these promoters plays a dominant role in controlling the relative levels of HLA-DRB1 and -DRB3 gene expression.