Localizing Transcriptional Regulatory Elements at the Mouse Dlk1 Locus

Much effort has focused recently on determining the mechanisms that control the allele-specific expression of genes subject to genomic imprinting, yet imprinting regulation is only one aspect of configuring appropriate expression of these genes. Imprinting control mechanisms must interact with those regulating the tissue-specific expression pattern of each imprinted gene in a cluster. Proper expression of the imprinted Delta-like 1 (Dlk1) - Maternally expressed gene 3 (Meg3) gene pair is required for normal fetal development in mammals, yet the mechanisms that control tissue-specific expression of these genes are unknown. We have used a combination of in vivo and in vitro expression assays to localize cis-regulatory elements that may regulate Dlk1 expression in the mouse embryo. A bacterial artificial chromosome transgene encompassing the Dlk1 gene and 77 kb of flanking sequence conferred expression in most endogenous Dlk1-expressing tissues. In combination with previous transgenic data, these experiments localize the majority of Dlk1 cis-regulatory elements to a 41 kb region upstream of the gene. Cross-species sequence conservation was used to further define potential regulatory elements, several of which functioned as enhancers in a luciferase expression assay. Two of these elements were able to drive expression of a lacZ reporter transgene in Dlk1-expressing tissues in the mouse embryo. The sequence proximal to Dlk1 therefore contains at least two discrete regions that may regulate tissue-specificity of Dlk1 expression

A multilevel examination of gender differences in the association between features of the school environment and physical activity among a sample of grades 9 to 12 students in Ontario, Canada

<p>Abstract</p> <p>Background</p> <p>Creating school environments that support student physical activity (PA) is a key recommendation of policy-makers to increase youth PA. Given males are more active than females at all ages, it has been suggested that investigating gender differences in the features of the environment that associate with PA may help to inform gender-focused PA interventions and reduce the gender disparity in PA. The purpose of this cross-sectional study was to explore gender differences in the association between factors of the school environment and students' time spent in PA.</p> <p>Methods</p> <p>Among a sample of 10781 female and 10973 male students in grades 9 to 12 from 76 secondary schools in Ontario, Canada, student- and school-level survey PA data were collected and supplemented with GIS-derived measures of the built environment within 1-km buffers of the 76 schools.</p> <p>Results</p> <p>Findings from the present study revealed significant differences in the time male and female students spent in PA as well as in some of the school- and student-level factors associated with PA. Results of the gender-specific multilevel analyses indicate schools should consider providing an alternate room for PA, especially for providing flexibility activities directed at female students. Schools should also consider offering daily physical education programming to male students in senior grades and providing PA promotion initiatives targeting obese male students.</p> <p>Conclusions</p> <p>Although most variation in male and female students' time spent in PA lies between students within schools, there is sufficient between-school variation to be of interest to practitioners and policy-makers. More research investigating gender differentials in environment factors associated with youth PA are warranted.</p

Genome-wide association and Mendelian randomisation analysis provide insights into the pathogenesis of heart failure

Heart failure (HF) is a leading cause of morbidity and mortality worldwide. A small proportion of HF cases are attributable to monogenic cardiomyopathies and existing genome-wide association studies (GWAS) have yielded only limited insights, leaving the observed heritability of HF largely unexplained. We report results from a GWAS meta-analysis of HF comprising 47,309 cases and 930,014 controls. Twelve independent variants at 11 genomic loci are associated with HF, all of which demonstrate one or more associations with coronary artery disease (CAD), atrial fibrillation, or reduced left ventricular function, suggesting shared genetic aetiology. Functional analysis of non-CAD-associated loci implicate genes involved in cardiac development (MYOZ1, SYNPO2L), protein homoeostasis (BAG3), and cellular senescence (CDKN1A). Mendelian randomisation analysis supports causal roles for several HF risk factors, and demonstrates CAD-independent effects for atrial fibrillation, body mass index, and hypertension. These findings extend our knowledge of the pathways underlying HF and may inform new therapeutic strategies

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p&lt;0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (&lt;1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (&lt;1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Genome-wide association and Mendelian randomisation analysis provide insights into the pathogenesis of heart failure

Abstract: Heart failure (HF) is a leading cause of morbidity and mortality worldwide. A small proportion of HF cases are attributable to monogenic cardiomyopathies and existing genome-wide association studies (GWAS) have yielded only limited insights, leaving the observed heritability of HF largely unexplained. We report results from a GWAS meta-analysis of HF comprising 47,309 cases and 930,014 controls. Twelve independent variants at 11 genomic loci are associated with HF, all of which demonstrate one or more associations with coronary artery disease (CAD), atrial fibrillation, or reduced left ventricular function, suggesting shared genetic aetiology. Functional analysis of non-CAD-associated loci implicate genes involved in cardiac development (MYOZ1, SYNPO2L), protein homoeostasis (BAG3), and cellular senescence (CDKN1A). Mendelian randomisation analysis supports causal roles for several HF risk factors, and demonstrates CAD-independent effects for atrial fibrillation, body mass index, and hypertension. These findings extend our knowledge of the pathways underlying HF and may inform new therapeutic strategies

Identification of Imprinting Regulators at the Meg3 Differentially Methylated Region

Genomic imprinting at the Delta-like 1 (Dlk1)-Maternally expressed gene 3 (Meg3) locus is regulated by the Meg3 differentially methylated region (DMR), but the mechanism by which this DMR acts is unknown. The goal of this study was to analyze the Meg3 DMR during imprinting establishment and maintenance for the presence of histone modifications and trans-acting DNA binding proteins using chromatin immunoprecipitation. In embryonic stem (ES) cells, where Meg3 is biallelically expressed, the DMR showed variable DNA methylation, with biallelic methylation at one region but paternal allele-specific methylation at another. All histone modifications detected at the Meg3 DMR of ES cells were biallelic. In embryonic day 12.5 (e12.5) embryos, where Meg3 is maternally expressed, the paternal Meg3 DMR was methylated, and activating histone modifications were specific to the maternal DMR. DNA-binding proteins that represent potential regulatory factors were identified in both ES cells and embryos

Embryonic expression of <i>CE4-lacZ</i>, <i>CE8-lacZ</i> and <i>Dlk1.</i>

<p>+ indicates expression; – indicates no expression.</p

Sequence conservation in the <i>Dlk1</i> upstream region.

<p>The sequence upstream of <i>Dlk1</i> was analyzed for regions of conservation across multiple species. The conserved elements chosen for further analysis are numbered CE1 to CE9. The region being displayed corresponds to the July 2007 mouse genome assembly; assembly dates for other species are given in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036483#s2" target="_blank">Methods</a> (adapted from the UCSC Genome Browser).</p

<i>CE4-lacZ</i> displays expression in a subset of <i>Dlk1</i>-expressing tissues.

<p>(A) Diagram of <i>lacZ</i> expression constructs used to produce <i>CE4-lacZ</i> and <i>CE8-lacZ</i> transgenic embryos. The arrow represents the direction of transcription, and CE stands for conserved element 4 or 8. (B–D) Ventral, dorsal and lateral views, respectively, of a representative whole mount transgenic embryo at e13.5. (E) Schematic representation of sagittal section planes used in these images <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036483#pone.0036483-Kaufman1" target="_blank">[64]</a>. (F–M) Sagittal sections of embryos under low magnification (F, I, L) and high magnification (G, H, J, K, M). All sections are oriented with anterior on top and dorsal to the left. Expression was seen in (F–H) intercostal muscle, body wall muscle, and ribs; (I, J) dorsal root ganglia; (I, K) intrinsic tongue muscle; (L, M) thymus. The embryo shown in (F) displays <i>lacZ</i> expression in the pituitary gland and trigeminal ganglion, both sites of endogenous <i>Dlk1</i> expression, but this pattern was not seen in other embryos carrying <i>CE4-lacZ</i>. Scale bars represent 100 µm.</p