Broadly expressed repressors integrate patterning across orthogonal axes in embryos

The role of spatially localized repressors in supporting embryonic patterning is well appreciated, but, alternatively, the role ubiquitously expressed repressors play in this process is not well understood. We investigated the function of two broadly expressed repressors, Runt (Run) and Suppressor of Hairless [Su(H)], in patterning the Drosophila embryo. Previous studies have shown that Run and Su(H) regulate gene expression along anterior-posterior (AP) or dorsal-ventral (DV) axes, respectively, by spatially limiting activator action, but here we characterize a different role. Our data show that broadly expressed repressors silence particular enhancers within cis-regulatory systems, blocking their expression throughout the embryo fully but transiently, and, in this manner, regulate spatiotemporal outputs along both axes. Our results suggest that Run and Su(H) regulate the temporal action of enhancers and are not dedicated regulators of one axis but, instead, act coordinately to pattern both axes, AP and DV

Distinct Roles of Broadly Expressed Repressors Support Dynamic Enhancer Action and Change in Time

How broadly expressed repressors regulate gene expression is incompletely understood. To gain insight, we investigated how Suppressor of Hairless—Su(H)—and Runt regulate expression of bone morphogenetic protein (BMP) antagonist short-gastrulation via the sog_Distal enhancer. A live imaging protocol was optimized to capture this enhancer’s spatiotemporal output throughout the early Drosophila embryo, finding in this context that Runt regulates transcription initiation, Su(H) regulates transcription rate, and both factors control spatial expression. Furthermore, whereas Su(H) functions as a dedicated repressor, Runt temporally switches from repressor to activator. Our results demonstrate that broad repressors play temporally distinct roles and contribute to dynamic gene expression. Both Run and Su(H)’s ability to influence the spatiotemporal domains of gene expression may serve to counterbalance activators and function in this manner as important regulators of the maternal-to-zygotic transition in early embryos

Odd-paired is a pioneer-like factor that coordinates with Zelda to control gene expression in embryos

Pioneer factors such as Zelda (Zld) help initiate zygotic transcription in Drosophila early embryos, but whether other factors support this dynamic process is unclear. Odd-paired (Opa), a zinc-finger transcription factor expressed at cellularization, controls the transition of genes from pair-rule to segmental patterns along the anterior-posterior axis. Finding that Opa also regulates expression through enhancer sog_Distal along the dorso-ventral axis, we hypothesized Opa’s role is more general. Chromatin-immunoprecipitation (ChIP-seq) confirmed its in vivo binding to sog_Distal but also identified widespread binding throughout the genome, comparable to Zld. Furthermore, chromatin assays (ATAC-seq) demonstrate that Opa, like Zld, influences chromatin accessibility genome-wide at cellularization, suggesting both are pioneer factors with common as well as distinct targets. Lastly, embryos lacking opa exhibit widespread, late patterning defects spanning both axes. Collectively, these data suggest Opa is a general timing factor and likely late-acting pioneer factor that drives a secondary wave of zygotic gene expression

Light-dependent N-end rule-mediated disruption of protein function in Saccharomyces cerevisiae and Drosophila melanogaster

Here we describe the development and characterization of the photo-N-degron, a peptide tag that can be used in optogenetic studies of protein function in vivo. The photo-N-degron can be expressed as a genetic fusion to the amino termini of other proteins, where it undergoes a blue light-dependent conformational change that exposes a signal for the class of ubiquitin ligases, the N-recognins, which mediate the N-end rule mechanism of proteasomal degradation. We demonstrate that the photo-N-degron can be used to direct light-mediated degradation of proteins in Saccharomyces cerevisiae and Drosophila melanogaster with fine temporal control. In addition, we compare the effectiveness of the photo-N-degron with that of two other light-dependent degrons that have been developed in their abilities to mediate the loss of function of Cactus, a component of the dorsal-ventral patterning system in the Drosophila embryo. We find that like the photo-N-degron, the blue light-inducible degradation (B-LID) domain, a light-activated degron that must be placed at the carboxy terminus of targeted proteins, is also effective in eliciting light-dependent loss of Cactus function, as determined by embryonic dorsal-ventral patterning phenotypes. In contrast, another previously described photosensitive degron (psd), which also must be located at the carboxy terminus of associated proteins, has little effect on Cactus-dependent phenotypes in response to illumination of developing embryos. These and other observations indicate that care must be taken in the selection and application of light-dependent and other inducible degrons for use in studies of protein function in vivo, but importantly demonstrate that N- and C-terminal fusions to the photo-N-degron and the B-LID domain, respectively, support light-dependent degradation in vivo

Cell-specific occupancy dynamics between the pioneer-like factor Opa/ZIC and Ocelliless/OTX regulate early head development in embryos

During development, embryonic patterning systems direct a set of initially uncommitted pluripotent cells to differentiate into a variety of cell types and tissues. A core network of transcription factors, such as Zelda/POU5F1, Odd-paired (Opa)/ZIC3 and Ocelliless (Oc)/OTX2, are conserved across animals. While Opa is essential for a second wave of zygotic activation after Zelda, it is unclear whether Opa drives head cell specification, in the Drosophila embryo. Our hypothesis is that Opa and Oc are interacting with distinct cis-regulatory regions for shaping cell fates in the embryonic head. Super-resolution microscopy and meta-analysis of single-cell RNAseq datasets show that opa’s and oc’s overlapping expression domains are dynamic in the head region, with both factors being simultaneously transcribed at the blastula stage. Additionally, analysis of single-embryo RNAseq data reveals a subgroup of Opa-bound genes to be Opa-independent in the cellularized embryo. Interrogation of these genes against Oc ChIPseq combined with in situ data, suggests that Opa is competing with Oc for the regulation of a subgroup of genes later in gastrulation. Specifically, we find that Oc binds to late, head-specific enhancers independently and activates them in a head-specific wave of zygotic transcription, suggesting distinct roles for Oc in the blastula and gastrula stages

A critical view of the general public's awareness and physicians' opinion of the trends and potential pitfalls of genetic testing in Greece

Aim: Progress in deciphering the functionality of the human genome sequence in the wake of technological advances in the field of genomic medicine have dramatically reduced the overall costs of genetic analysis, thereby facilitating the incorporation of genetic testing services into mainstream clinical practice. Although Greek genetic testing laboratories offer a variety of different genetic tests, relatively little is known about how either the general public or medical practitioners perceive genetic testing services. Materials & methods: We have therefore performed a nationwide survey of the views of 1717 members of the general public, divided into three age groups, from all over Greece, and residing in both large and small cities and villages, in order to acquire a better understanding of how they perceive genetic testing. We also canvassed the opinions of 496 medical practitioners with regard to genetic testing services in a separate survey that addressed similar issues. Results: Our subsequent analysis indicated that a large proportion of the general public is aware of the nature of DNA, genetic disorders and the potential benefits of genetic testing, although this proportion declines steadily with age. Furthermore, a large proportion of the interviewed individuals would be willing to undergo genetic testing even if the cost of analysis was not covered by healthcare insurance. However, a relatively small proportion of the general public has actually been advized to undergo genetic testing, either by relatives or physicians. Most physicians believe that the regulatory and legal framework that governs genetic testing services in Greece is rather weak. Interestingly, the vast majority of the general public strongly opposes direct-access genetic testing, and most would prefer referral from a physician than from a pharmacist. Conclusion: Overall, our results provide a critical evaluation of the views of the general public with regard to genetics and genetic testing services in Greece and should serve as a model for replication in other populations

Assessment of gene-by-sex interaction effect on bone mineral density

To access publisher's full text version of this article. Please click on the hyperlink in Additional Links field.Sexual dimorphism in various bone phenotypes, including bone mineral density (BMD), is widely observed; however, the extent to which genes explain these sex differences is unclear. To identify variants with different effects by sex, we examined gene-by-sex autosomal interactions genome-wide, and performed expression quantitative trait loci (eQTL) analysis and bioinformatics network analysis. We conducted an autosomal genome-wide meta-analysis of gene-by-sex interaction on lumbar spine (LS) and femoral neck (FN) BMD in 25,353 individuals from 8 cohorts. In a second stage, we followed up the 12 top single-nucleotide polymorphisms (SNPs; p < 1 × 10(-5) ) in an additional set of 24,763 individuals. Gene-by-sex interaction and sex-specific effects were examined in these 12 SNPs. We detected one novel genome-wide significant interaction associated with LS-BMD at the Chr3p26.1-p25.1 locus, near the GRM7 gene (male effect = 0.02 and p = 3.0 × 10(-5) ; female effect = -0.007 and p = 3.3 × 10(-2) ), and 11 suggestive loci associated with either FN- or LS-BMD in discovery cohorts. However, there was no evidence for genome-wide significant (p < 5 × 10(-8) ) gene-by-sex interaction in the joint analysis of discovery and replication cohorts. Despite the large collaborative effort, no genome-wide significant evidence for gene-by-sex interaction was found to influence BMD variation in this screen of autosomal markers. If they exist, gene-by-sex interactions for BMD probably have weak effects, accounting for less than 0.08% of the variation in these traits per implicated SNP. © 2012 American Society for Bone and Mineral Research.Medtronic NIH R01 AG18728 R01HL088119 R01AR046838 U01 HL084756 R01 AR43351 P01-HL45522 R01-MH-078111 R01-MH-083824 Nutrition and Obesity Research Center of Maryland P30DK072488 NIAMS/NIH F32AR059469 Instituto de Salud Carlos III-FIS (Spanish Health Ministry) PI 06/0034 PI08/0183 Canadian Institutes of Health Research (CIHR) NHLBI HHSN268201200036C N01-HC-85239 N01-HC-85079 N01-HC-85086 N01-HC-35129 N01 HC15103 N01 HC-55222 N01-HC-75150 N01-HC-45133 HL080295 HL087652 HL105756 NIA AG-023629 AG-15928 AG-20098 AG-027058 N01AG62101 N01AG62103 N01AG62106 1R01AG032098-01A1 National Center of Advancing Translational Technologies CTSI UL1TR000124 National Institute of Diabetes and Digestive and Kidney Diseases DK063491 EUROSPAN (European Special Populations Research Network) European Commission FP6 STRP grant 018947 LSHG-CT-2006-01947 Netherlands Organisation for Scientific Research Erasmus MC Centre for Medical Systems Biology (CMSB) Netherlands Brain Foundation (HersenStichting Nederland) US National Institute for Arthritis, Musculoskeletal and Skin Diseases National Institute on Aging R01 AR/AG41398 R01 AR050066 R21 AR056405 National Heart, Lung, and Blood Institute's Framingham Heart Study N01-HC-25195 Affymetrix, Inc. N02-HL-6-4278 Canadian Institutes of Health Research from Institute of Aging 165446 Institute of Genetics 179433 Institute of Musculoskeletal health 221765 Intramural Research Program of the NIH, National Institute on Aging National Institutes of Health HHSN268200782096C Hong Kong Research Grant Council HKU 768610M Bone Health Fund of HKU Foundation KC Wong Education Foundation Small Project Funding 201007176237 Matching Grant CRCG Grant Osteoporosis and Endocrine Research Fund Genomics Strategic Research Theme of The University of Hong Kong Netherlands Organisation of Scientific Research NWO Investments 175.010.2005.011 911-03-012 Research Institute for Diseases in the Elderly 014-93-015 Netherlands Genomics Initiative (NGI)/Netherlands Consortium for Healthy Aging (NCHA) 050-060-810 Erasmus Medical Center and Erasmus University, Rotterdam Netherlands Organization for the Health Research and Development (ZonMw) Research Institute for Diseases in the Elderly (RIDE) Ministry of Education, Culture and Science Ministry for Health, Welfare and Sports European Commission (DG XII) Municipality of Rotterdam German Bundesministerium fur Forschung und Technology 01 AK 803 A-H 01 IG 07015

Genetic Sharing with Cardiovascular Disease Risk Factors and Diabetes Reveals Novel Bone Mineral Density Loci.

Bone Mineral Density (BMD) is a highly heritable trait, but genome-wide association studies have identified few genetic risk factors. Epidemiological studies suggest associations between BMD and several traits and diseases, but the nature of the suggestive comorbidity is still unknown. We used a novel genetic pleiotropy-informed conditional False Discovery Rate (FDR) method to identify single nucleotide polymorphisms (SNPs) associated with BMD by leveraging cardiovascular disease (CVD) associated disorders and metabolic traits. By conditioning on SNPs associated with the CVD-related phenotypes, type 1 diabetes, type 2 diabetes, systolic blood pressure, diastolic blood pressure, high density lipoprotein, low density lipoprotein, triglycerides and waist hip ratio, we identified 65 novel independent BMD loci (26 with femoral neck BMD and 47 with lumbar spine BMD) at conditional FDR < 0.01. Many of the loci were confirmed in genetic expression studies. Genes validated at the mRNA levels were characteristic for the osteoblast/osteocyte lineage, Wnt signaling pathway and bone metabolism. The results provide new insight into genetic mechanisms of variability in BMD, and a better understanding of the genetic underpinnings of clinical comorbidity

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms