The promoter polymorphism -232C/G of the PCK1 gene is associated with type 2 diabetes in a UK-resident South Asian population

Background - The PCK1 gene, encoding cytosolic phosphoenolpyruvate carboxykinase (PEPCKC), has previously been implicated as a candidate gene for type 2 diabetes (T2D) susceptibility. Rodent models demonstrate that over-expression of Pck1 can result in T2D development and a single nucleotide polymorphism (SNP) in the promoter region of human PCK1 (-232C/G) has exhibited significant association with the disease in several cohorts. Within the UK-resident South Asian population, T2D is 4 to 6 times more common than in indigenous white Caucasians. Despite this, few studies have reported on the genetic susceptibility to T2D in this ethnic group and none of these has investigated the possible effect of PCK1 variants. We therefore aimed to investigate the association between common variants of the PCK1 gene and T2D in a UK-resident South Asian population of Punjabi ancestry, originating predominantly from the Mirpur area of Azad Kashmir, Pakistan. Methods - We used TaqMan assays to genotype five tagSNPs covering the PCK1 gene, including the -232C/G variant, in 903 subjects with T2D and 471 normoglycaemic controls. Results - Of the variants studied, only the minor allele (G) of the -232C/G SNP demonstrated a significant association with T2D, displaying an OR of 1.21 (95% CI: 1.03 - 1.42, p = 0.019). Conclusion - This study is the first to investigate the association between variants of the PCK1 gene and T2D in South Asians. Our results suggest that the -232C/G promoter polymorphism confers susceptibility to T2D in this ethnic group.

Selective inhibition of the human tie-1 promoter with triplex-forming oligonucleotides targeted to ets binding sites

The Tie receptors (Tie-1 and Tie-2/Tek) are essential for angiogenesis and vascular remodeling/integrity. Tie receptors are up-regulated in tumor-associated endothelium, and their inhibition disrupts angiogenesis and can prevent tumor growth as a consequence. To investigate the potential of anti-gene approaches to inhibit tie gene expression for anti-angiogenic therapy, we have examined triple-helical (triplex) DNA formation at 2 tandem Ets transcription factor binding motifs (designated E-1 and E-2) in the human tie-1 promoter. Various tie-1 promoter deletion/mutation luciferase reporter constructs were generated and transfected into endothelial cells to examine the relative activities of E-1 and E-2. The binding of antiparallel and parallel (control) purine motif oligonucleotides (21-22 bp) targeted to E-1 and E-2 was assessed by plasmid DNA fragment binding and electrophoretic mobility shift assays. Triplex-forming oligonucleotides were incubated with tie-1 reporter constructs and transfected into endothelial cells to determine their activity. The Ets binding motifs in the E-1 sequence were essential for human tie-1 promoter activity in endothelial cells, whereas the deletion of E-2 had no effect. Antiparallel purine motif oligonucleotides targeted at E-1 or E-2 selectively formed strong triplex DNA (K(d) approximately 10(-7) M) at 37 degrees C. Transfection of tie-1 reporter constructs with triplex DNA at E-1, but not E-2, specifically inhibited tie-1 promoter activity by up to 75% compared with control oligonucleotides in endothelial cells. As similar multiple Ets binding sites are important for the regulation of several endothelial-restricted genes, this approach may have broad therapeutic potential for cancer and other pathologies involving endothelial proliferation/dysfunction.

Promoter effects in the polymerisation of a mixed hydrocarbon feed with silica-supported BF3

The activity of a silica-supported BF3–methanol solid acid catalyst in the cationic polymerisation of an industrial aromatic C9 feedstock has been investigated. Reuse has been achieved under continuous conditions. Titration of the catalyst acid sites with triethylphosphine oxide (TEPO) in conjunction with 31P MAS NMR shows the catalyst to have two types of acid sites. Further analysis with 2,6 di-tert-butyl-4-methylpyridine (DBMP) has revealed the majority of these acid sites to be Brønsted in nature. The role of α-methylstyrene in promoting resin polymerisation via chain transfer is proposed.

Derivation of Context-free Stochastic L-Grammar Rules for Promoter Sequence Modeling Using Support Vector Machine

Formal grammars can used for describing complex repeatable structures such as DNA sequences. In this paper, we describe the structural composition of DNA sequences using a context-free stochastic L-grammar. L-grammars are a special class of parallel grammars that can model the growth of living organisms, e.g. plant development, and model the morphology of a variety of organisms. We believe that parallel grammars also can be used for modeling genetic mechanisms and sequences such as promoters. Promoters are short regulatory DNA sequences located upstream of a gene. Detection of promoters in DNA sequences is important for successful gene prediction. Promoters can be recognized by certain patterns that are conserved within a species, but there are many exceptions which makes the promoter recognition a complex problem. We replace the problem of promoter recognition by induction of context-free stochastic L-grammar rules, which are later used for the structural analysis of promoter sequences. L-grammar rules are derived automatically from the drosophila and vertebrate promoter datasets using a genetic programming technique and their fitness is evaluated using a Support Vector Machine (SVM) classifier. The artificial promoter sequences generated using the derived L- grammar rules are analyzed and compared with natural promoter sequences.

The handedness-associated PCSK6 locus spans an intronic promoter regulating novel transcripts

We recently reported the association of the PCSK6 gene with handedness through a quantitative genome-wide association study (GWAS; P < 0.5 × 10(-8)) for a relative hand skill measure in individuals with dyslexia. PCSK6 activates Nodal, a morphogen involved in regulating left-right body axis determination. Therefore, the GWAS data suggest that the biology underlying the patterning of structural asymmetries may also contribute to behavioural laterality, e.g. handedness. The association is further supported by an independent study reporting a variable number tandem repeat (VNTR) within the same PCSK6 locus to be associated with degree of handedness in a general population cohort. Here, we have conducted a functional analysis of the PCSK6 locus combining further genetic analysis, in silico predictions and molecular assays. We have shown that the previous GWAS signal was not tagging a VNTR effect, suggesting that the two markers have independent effects. We demonstrated experimentally that one of the top GWAS-associated markers, rs11855145, directly alters the binding site for a nuclear factor. Furthermore, we have shown that the predicted regulatory region adjacent to rs11855415 acts as a bidirectional promoter controlling the expression of novel RNA transcripts. These include both an antisense long non-coding RNA (lncRNA) and a short PCSK6 isoform predicted to be coding. This is the first molecular characterization of a handedness-associated locus that supports the role of common variants in non-coding sequences in influencing complex phenotypes through gene expression regulation.

Analysis of the effects of depression associated polymorphisms on the activity of the BICC1 promoter in amygdala neurones

ACKNOWLEDGMENTS This work was funded by The BBSRC (BB/D004659/1) the Wellcome Trust (080980/Z/06/Z) and the Medical Research Council (G0701003).

The human preprotachykinin-A gene promoter has been highly conserved and can drive human-like marker gene expression in the adult mouse CNS.

Peer reviewed

The human preprotachykinin-A gene promoter has been highly conserved and can drive human-like marker gene expression in the adult mouse CNS.

Peer reviewed

Maternal and paternal growth regulatory factor expression in hybrid sunfish.

Unidirectional hybridization between bluegill (Lepomis macrochirus) and pumpkinseed (L. gibbosus) sunfish enables researchers to explore the relative expression of paternal and maternal alleles in hybrids. Past studies have found that the metabolic dysfunction in bluegill-pumpkinseed hybrids may be due to incompatibilities between nuclear and mitochondrial genomes. However, the consequences of hybridization on body size and muscle growth have not been examined. This topic is particularly interesting because hybrids grow larger than parentals despite the fact that they are often sired by smaller, precociously mature bluegills. In order to improve our understanding of growth dynamics in hybrid sunfish, I conducted real-time quantitative PCR using species-specific primers on the white muscle tissue of bluegills, pumpkinseeds, and hybrids collected from Lake Opinicon, ON. Five growth factors that have been linked to muscle growth and body size demonstrated similar expression for maternal and paternal alleles. While about half of the hybrids showed the same pattern with myogenin, about half showed very low levels of mRNA for the paternal (bluegill) gene. While this did not explain the heterosis seen in hybrids, it may explain the small body phenotype of the cuckholding bluegill males. I explored the upstream genetic structure of bluegill myogenin and established that four alleles exist within the population. Furthermore, I uncovered a relationship in hybrids between the proximal promoter/ 5’ UTR of myogenin and its transcript level. I found that the hybrids demonstrating low paternal myogenin expression unfailingly possessed A3 or A4 alleles, but future studies will be needed to reveal the molecular links between the genotype and the growth phenotype. A similar genotype-phenotype association was not obvious in parentals, even those that were homozygous for these alleles. Whether this relationship can provide insight into the genetic determinants of bluegill alternative mating strategies has yet to be determined.

Hypoxia-induced DNA hypermethylation in human pulmonary fibroblasts is associated with Thy-1 promoter methylation and the development of a pro-fibrotic phenotype

BACKGROUND: Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression.

METHODS: CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter.

RESULTS: Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-2'-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2.

CONCLUSION: These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia.