The human initiator is a distinct and abundant element that is precisely positioned in focused core promoters.

DNA sequence signals in the core promoter, such as the initiator (Inr), direct transcription initiation by RNA polymerase II. Here we show that the human Inr has the consensus of BBCA+1BW at focused promoters in which transcription initiates at a single site or a narrow cluster of sites. The analysis of 7678 focused transcription start sites revealed 40% with a perfect match to the Inr and 16% with a single mismatch outside of the CA+1 core. TATA-like sequences are underrepresented in Inr promoters. This consensus is a key component of the DNA sequence rules that specify transcription initiation in humans

High throughput mutagenesis for identification of residues regulating human prostacyclin (hIP) receptor

The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structurefunction relationship of GPCRs. © 2014 Bill et al

Relationship between smoking and obesity:a cross-sectional study of 499,504 middle-aged adults in the UK general population

Background: There is a general perception that smoking protects against weight gain and this may influence commencement and continuation of smoking, especially among young women.<p></p> Methods: A cross-sectional study was conducted using baseline data from UK Biobank. Logistic regression analyses were used to explore the association between smoking and obesity; defined as body mass index (BMI) >30kg/m2. Smoking was examined in terms of smoking status, amount smoked, duration of smoking and time since quitting and we adjusted for the potential confounding effects of age, sex, socioeconomic deprivation, physical activity, alcohol consumption, hypertension and diabetes.<p></p> Results: The study comprised 499,504 adults aged 31 to 69 years. Overall, current smokers were less likely to be obese than never smokers (adjusted OR 0.83 95% CI 0.81-0.86). However, there was no significant association in the youngest sub-group (≤40 years). Former smokers were more likely to be obese than both current smokers (adjusted OR 1.33 95% CI 1.30-1.37) and never smokers (adjusted OR 1.14 95% CI 1.12-1.15). Among smokers, the risk of obesity increased with the amount smoked and former heavy smokers were more likely to be obese than former light smokers (adjusted OR 1.60, 95% 1.56-1.64, p<0.001). Risk of obesity fell with time from quitting. After 30 years, former smokers still had higher risk of obesity than current smokers but the same risk as never smokers.<p></p> Conclusion: Beliefs that smoking protects against obesity may be over-simplistic; especially among younger and heavier smokers. Quitting smoking may be associated with temporary weight gain. Therefore, smoking cessation interventions should include weight management support.<p></p&gt

CYP17 5'-UTR MspA1 polymorphism and the risk of premenopausal breast cancer in a German population-based case–control study

INTRODUCTION: Studies on the association between the cytochrome P450c17α gene (CYP17) 5'-untranslated region MspA1 genetic polymorphism and breast cancer risk have yielded inconsistent results. Higher levels of estrogen have been reported among young nulliparous women with the A2 allele. Therefore we assessed the impact of CYP17 genotypes on the risk of premenopausal breast cancer, with emphasis on parity. METHODS: We used data from a population-based case–control study of women aged below 51 years conducted from 1992 to 1995 in Germany. Analyses were restricted to clearly premenopausal women with complete information on CYP17 and encompassed 527 case subjects and 904 controls, 99.5% of whom were of European descent. The MspA1 polymorphism was analyzed using PCR-RFLP (PCR–restriction fragment length polymorphism) assay. RESULTS: The frequencies of the variant allele among the cases and controls were 43% and 41%, respectively. Overall, CYP17 A1/A2 and A2/A2 genotypes compared with the A1/A1 genotype were not associated with breast cancer, with adjusted odds ratios (ORs) of 1.04 and 1.23, respectively. Among nulliparous women, however, breast cancer risk was elevated for the A1/A2 (OR = 1.31; 95% confidence interval (CI) 0.74 to 2.32) and the A2/A2 genotype (OR = 2.12; 95% CI 1.04 to 4.32) compared with the A1/A1 genotype, with a trend towards increasing risk associated with number of A2 alleles (P = 0.04). Otherwise, the CYP17 polymorphism was found neither to be an effect modifier of breast cancer risks nor to be associated with stage of disease. CONCLUSION: Our results do not indicate a major influence of CYP17 MspA1 polymorphism on the risk of premenopausal breast cancer, but suggest that it may have an impact on breast cancer risk among nulliparous women. The finding, however, needs to be confirmed in further studies

How to Build Transcriptional Network Models of Mammalian Pattern Formation

Genetic regulatory networks of sequence specific transcription factors underlie pattern formation in multicellular organisms. Deciphering and representing the mammalian networks is a central problem in development, neurobiology, and regenerative medicine. Transcriptional networks specify intermingled embryonic cell populations during pattern formation in the vertebrate neural tube. Each embryonic population gives rise to a distinct type of adult neuron. The homeodomain transcription factor Lbx1 is expressed in five such populations and loss of Lbx1 leads to distinct respecifications in each of the five populations. allele, respectively. Microarrays were used to show that expression levels of 8% of all transcription factor genes were altered in the respecified pool. These transcription factor genes constitute 20–30% of the active nodes of the transcriptional network that governs neural tube patterning. Half of the 141 regulated nodes were located in the top 150 clusters of ultraconserved non-coding regions. Generally, Lbx1 repressed genes that have expression patterns outside of the Lbx1-expressing domain and activated genes that have expression patterns inside the Lbx1-expressing domain.nalysis, and think that it will be generally useful in discovering and assigning network interactions to specific populations. We discuss how ANCEA, coupled with population partitioning analysis, can greatly facilitate the systematic dissection of transcriptional networks that underlie mammalian patterning

Microarray Profiling of Phage-Display Selections for Rapid Mapping of Transcription Factor–DNA Interactions

Modern computational methods are revealing putative transcription-factor (TF) binding sites at an extraordinary rate. However, the major challenge in studying transcriptional networks is to map these regulatory element predictions to the protein transcription factors that bind them. We have developed a microarray-based profiling of phage-display selection (MaPS) strategy that allows rapid and global survey of an organism's proteome for sequence-specific interactions with such putative DNA regulatory elements. Application to a variety of known yeast TF binding sites successfully identified the cognate TF from the background of a complex whole-proteome library. These factors contain DNA-binding domains from diverse families, including Myb, TEA, MADS box, and C2H2 zinc-finger. Using MaPS, we identified Dot6 as a trans-active partner of the long-predicted orphan yeast element Polymerase A & C (PAC). MaPS technology should enable rapid and proteome-scale study of bi-molecular interactions within transcriptional networks

Novel Allelic Variants in the Canine Cyclooxgenase-2 (Cox-2) Promoter Are Associated with Renal Dysplasia in Dogs

Renal dysplasia (RD) in dogs is a complex disease with a highly variable phenotype and mode of inheritance that does not follow a simple Mendelian pattern. Cox-2 (Cyclooxgenase-2) deficient mice have renal abnormalities and a pathology that has striking similarities to RD in dogs suggesting to us that mutations in the Cox-2 gene could be the cause of RD in dogs. Our data supports this hypothesis. Sequencing of the canine Cox-2 gene was done from clinically affected and normal dogs. Although no changes were detected in the Cox-2 coding region, small insertions and deletions of GC boxes just upstream of the ATG translation start site were found. These sequences are putative SP1 transcription factor binding sites that may represent important cis-acting DNA regulatory elements that govern the expression of Cox-2. A pedigree study of a family of Lhasa apsos revealed an important statistical correlation of these mutant alleles with the disease. We examined an additional 22 clinical cases from various breeds. Regardless of the breed or severity of disease, all of these had one or two copies of the Cox-2 allelic variants. We suggest that the unusual inheritance pattern of RD is due to these alleles, either by changing the pattern of expression of Cox-2 or making Cox-2 levels susceptible to influences of other genes or environmental factors that play an unknown but important role in the development of RD in dogs