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Mitochondria function as metabolic powerhouses by generating energy through oxidative phosphorylation and have become the focus of renewed interest due to progress in understanding the subtleties of their biogenesis and the discovery of the important roles which these organelles play in senescence, cell death and the assembly of iron-sulfur (Fe/S) centers. Using proteins from the yeast Saccharomyces cerevisiae, Homo sapiens and Arabidopsis thaliana we searched the sugarcane expressed sequence tag (SUCEST) database for the presence of expressed sequence tags (ESTs) with similarity to nuclear genes related to mitochondrial functions. Starting with 869 protein sequences, we searched for sugarcane EST counterparts to these proteins using the basic local alignment search tool TBLASTN similarity searching program run against 260,781 sugarcane ESTs contained in 81,223 clusters. We were able to recover 367 clusters likely to represent sugarcane orthologues of the corresponding genes from S. cerevisiae, H. sapiens and A. thaliana with E-value

Paternal diet can influence the phenotype of the next generation, yet, the dietary components inducing specific responses in the offspring are not identified. Here, we use the Nutritional Geometry Framework to determine the effects of pre-conception paternal dietary macronutrient balance on offspring metabolic and behavioral traits in mice. Ten isocaloric diets varying in the relative proportion of protein, fats, and carbohydrates are fed to male mice prior to mating. Dams and offspring are fed standard chow and never exposed to treatment diets. Body fat in female offspring is positively associated with the paternal consumption of fat, while in male offspring, an anxiety-like phenotype is associated to paternal diets low in protein and high in carbohydrates. Our study uncovers that the nature and the magnitude of paternal effects are driven by interactions between macronutrient balance and energy intake and are not solely the result of over- or undernutrition., (© 2024. The Author(s).)

Vascular homeostasis and pathophysiology are tightly regulated by mechanical forces generated by hemodynamics. Vascular disorders such as atherosclerotic diseases largely occur at curvatures and bifurcations where disturbed blood flow activates endothelial cells while unidirectional flow at the straight part of vessels promotes endothelial health. Integrated analysis of the endothelial transcriptome, the 3D epigenome, and human genetics systematically identified the SNP-enriched cistrome in vascular endothelium subjected to well-defined atherosclerosis-prone disturbed flow or atherosclerosis-protective unidirectional flow. Our results characterized the endothelial typical- and super-enhancers and underscored the critical regulatory role of flow-sensitive endothelial super-enhancers. CRISPR interference and activation validated the function of a previously unrecognized unidirectional flow-induced super-enhancer that upregulates antioxidant genes NQO1, CYB5B, and WWP2, and a disturbed flow-induced super-enhancer in endothelium which drives prothrombotic genes EDN1 and HIVEP in vascular endothelium. Our results employing multiomics identify the cis-regulatory architecture of the flow-sensitive endothelial epigenome related to atherosclerosis and highlight the regulatory role of super-enhancers in mechanotransduction mechanisms., (© 2024 Li et al.)

Diet is a key lifestyle component that influences metabolic health through several factors, including total energy intake and macronutrient composition. While the impact of caloric intake on gene expression and physiological phenomena in various tissues is well described, the influence of dietary macronutrient composition on these parameters is less well studied. Here, we use the Nutritional Geometry framework to investigate the role of macronutrient composition on metabolic function and gene regulation in adipose tissue. Using ten isocaloric diets that vary systematically in their proportion of energy from fat, protein, and carbohydrates, we find that gene expression and splicing are highly responsive to macronutrient composition, with distinct sets of genes regulated by different macronutrient interactions. Specifically, the expression of many genes associated with Bardet-Biedl syndrome is responsive to dietary fat content. Splicing and expression changes occur in largely separate gene sets, highlighting distinct mechanisms by which dietary composition influences the transcriptome and emphasizing the importance of considering splicing changes to more fully capture the gene regulation response to environmental changes such as diet. Our study provides insight into the gene regulation plasticity of adipose tissue in response to macronutrient composition, beyond the already well-characterized response to caloric intake., (© 2024. The Author(s).)

The Ras GTPase-activating protein SYNGAP1 plays a central role in synaptic plasticity, and de novo SYNGAP1 mutations are among the most frequent causes of autism and intellectual disability. How SYNGAP1 is regulated during development and how to treat SYNGAP1-associated haploinsufficiency remain challenging questions. Here, we characterize an alternative 3' splice site (A3SS) of SYNGAP1 that induces nonsense-mediated mRNA decay (A3SS-NMD) in mouse and human neural development. We demonstrate that PTBP1/2 directly bind to and promote SYNGAP1 A3SS inclusion. Genetic deletion of the Syngap1 A3SS in mice upregulates Syngap1 protein and alleviates the long-term potentiation and membrane excitability deficits caused by a Syngap1 knockout allele. We further report a splice-switching oligonucleotide (SSO) that converts SYNGAP1 unproductive isoform to the functional form in human iPSC-derived neurons. This study describes the regulation and function of SYNGAP1 A3SS-NMD, the genetic rescue of heterozygous Syngap1 knockout mice, and the development of an SSO to potentially alleviate SYNGAP1-associated haploinsufficiency., Competing Interests: Declaration of interests A provisional patent on splice-switching oligonucleotides described in this study has been filed on behalf of X.Z. and R.Y., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The mechanisms that underlie the timing of labor in humans are largely unknown. In most pregnancies, labor is initiated at term (≥ 37 weeks gestation), but in a signifiicant number of women spontaneous labor occurs preterm and is associated with increased perinatal mortality and morbidity. The objective of this study was to characterize the cells at the maternal-fetal interface (MFI) in term and preterm pregnancies in both the laboring and non-laboring state in Black women, who have among the highest preterm birth rates in the U.S. Using mass cytometry to obtain high-dimensional single-cell resolution, we identified 31 cell populations at the MFI, including 25 immune cell types and six non-immune cell types. Among the immune cells, maternal PD1 + CD8 T cell subsets were less abundant in term laboring compared to term non-laboring women. Among the non-immune cells, PD-L1 + maternal (stromal) and fetal (extravillous trophoblast) cells were less abundant in preterm laboring compared to term laboring women. Consistent with these observations, the expression of CD274, the gene encoding PD-L1, was significantly depressed and less responsive to fetal signaling molecules in cultured mesenchymal stromal cells from the decidua of preterm compared to term women. Overall, these results suggest that the PD1/PD-L1 pathway at the MFI may perturb the delicate balance between immune tolerance and rejection and contribute to the onset of spontaneous preterm labor., (© 2023. The Author(s).)

Dietary factors influence male reproductive function in both experimental and epidemiological studies. However, there are currently no specific dietary guidelines for male preconception health. Here, we use the Nutritional Geometry framework to examine the effects of dietary macronutrient balance on reproductive traits in C57BL/6 J male mice. Dietary effects are observed in a range of morphological, testicular and spermatozoa traits, although the relative influence of protein, fat, carbohydrate, and their interactions differ depending on the trait being examined. Interestingly, dietary fat has a positive influence on sperm motility and antioxidant capacity, differing to typical high fat diet studies where calorie content is not controlled for. Moreover, body adiposity is not significantly correlated with any of the reproductive traits measured in this study. These results demonstrate the importance of macronutrient balance and calorie intake on reproductive function and support the need to develop specific, targeted, preconception dietary guidelines for males., (© 2023. The Author(s).)

Recent large-scale genomic association studies found evidence for a genetic link between increased risk of type 2 diabetes and decreased risk for adiposity-related traits, reminiscent of metabolically obese normal weight (MONW) association signatures. However, the target genes and cellular mechanisms driving such MONW associations remain to be identified. Here, we systematically identify the cellular programmes of one of the top-scoring MONW risk loci, the 2q24.3 risk locus, in subcutaneous adipocytes. We identify a causal genetic variant, rs6712203, an intronic single-nucleotide polymorphism in the COBLL1 gene, which changes the conserved transcription factor motif of POU domain, class 2, transcription factor 2, and leads to differential COBLL1 gene expression by altering the enhancer activity at the locus in subcutaneous adipocytes. We then establish the cellular programme under the genetic control of the 2q24.3 MONW risk locus and the effector gene COBLL1, which is characterized by impaired actin cytoskeleton remodelling in differentiating subcutaneous adipocytes and subsequent failure of these cells to accumulate lipids and develop into metabolically active and insulin-sensitive adipocytes. Finally, we show that perturbations of the effector gene Cobll1 in a mouse model result in organismal phenotypes matching the MONW association signature, including decreased subcutaneous body fat mass and body weight along with impaired glucose tolerance. Taken together, our results provide a mechanistic link between the genetic risk for insulin resistance and low adiposity, providing a potential therapeutic hypothesis and a framework for future identification of causal relationships between genome associations and cellular programmes in other disorders., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Introduction: Variants in the tau gene (MAPT) region are associated with breast cancer in women and Alzheimer's disease (AD) among persons lacking apolipoprotein E ε4 (ε4-)., Methods: To identify novel genes associated with tau-related pathology, we conducted two genome-wide association studies (GWAS) for AD, one among 10,340 ε4- women in the Alzheimer's Disease Genetics Consortium (ADGC) and another in 31 members (22 women) of a consanguineous Hutterite kindred., Results: We identified novel associations of AD with MGMT variants in the ADGC (rs12775171, odds ratio [OR] = 1.4, P = 4.9 × 10 -8 ) and Hutterite (rs12256016 and rs2803456, OR = 2.0, P = 1.9 × 10 -14 ) datasets. Multi-omics analyses showed that the most significant and largest number of associations among the single nucleotide polymorphisms (SNPs), DNA-methylated CpGs, MGMT expression, and AD-related neuropathological traits were observed among women. Furthermore, promoter capture Hi-C analyses revealed long-range interactions of the MGMT promoter with MGMT SNPs and CpG sites., Discussion: These findings suggest that epigenetically regulated MGMT expression is involved in AD pathogenesis, especially in women., (© 2022 The Authors. Alzheimer's & Dementia published by Wiley Periodicals LLC on behalf of Alzheimer's Association.)

Background: Asthma is the most common chronic disease in children, occurring at higher frequencies and with more severe disease in children with African ancestry., Methods: We tested for association with haplotypes at the most replicated and significant childhood-onset asthma locus at 17q12-q21 and asthma in European American and African American children. Following this, we used whole-genome sequencing data from 1060 African American and 100 European American individuals to identify novel variants on a high-risk African American-specific haplotype. We characterized these variants in silico using gene expression and ATAC-seq data from airway epithelial cells, functional annotations from ENCODE, and promoter capture (pc)Hi-C maps in airway epithelial cells. Candidate causal variants were then assessed for correlation with asthma-associated phenotypes in African American children and adults., Results: Our studies revealed nine novel African-specific common variants, enriched on a high-risk asthma haplotype, which regulated the expression of GSDMA in airway epithelial cells and were associated with features of severe asthma. Using ENCODE annotations, ATAC-seq, and pcHi-C, we narrowed the associations to two candidate causal variants that are associated with features of T2 low severe asthma., Conclusions: Previously unknown genetic variation at the 17q12-21 childhood-onset asthma locus contributes to asthma severity in individuals with African ancestries. We suggest that many other population-specific variants that have not been discovered in GWAS contribute to the genetic risk for asthma and other common diseases., (© 2022. The Author(s).)

Various human diseases and pregnancy-related disorders reflect endometrial dysfunction. However, rodent models do not share fundamental biological processes with the human endometrium, such as spontaneous decidualization, and no existing human cell cultures recapitulate the cyclic interactions between endometrial stromal and epithelial compartments necessary for decidualization and implantation. Here we report a protocol differentiating human pluripotent stem cells into endometrial stromal fibroblasts (PSC-ESFs) that are highly pure and able to decidualize. Coculture of PSC-ESFs with placenta-derived endometrial epithelial cells generated organoids used to examine stromal-epithelial interactions. Cocultures exhibited specific endometrial markers in the appropriate compartments, organization with cell polarity, and hormone responsiveness of both cell types. Furthermore, cocultures recapitulate a central feature of the human decidua by cyclically responding to hormone withdrawal followed by hormone retreatment. This advance enables mechanistic studies of the cyclic responses that characterize the human endometrium., Competing Interests: Declaration of interests V.C.C., C.-Y.P., and J.A.K. have filed a provisional patent for the protocol described. M.M., N.J.S., I.A., M.A.N., C.O., and V.J.L. declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Sewage sludge (SS), a solid residue of effluent treatment, is rich in organic matter and nutrients, while also containing heavy metals and other potential contaminants. The feasibility of employing SS as a substrate component for seedling production depends on its composition and dose, as well as on the tolerance limit of individual plant species. To expand the knowledge base on the use of SS in the production of plants native to the Brazilian Cerrado biome, we evaluated the physiological responses and quality of Alibertia edulis seedlings grown under distinct SS concentrations. Chlorophyll a fluorescence (ChlF), stomatal conductance (gs), leaf temperature (Lt), biomass, growth, and seedling quality were investigated. At 25%, SS improved growth, biomass, and seedling quality, while substrates containing 50% or more SS affected gs, Lt, and ChlF. Seedling quality was strongly worsened by SS at 75% and 100%, as shown by lower biomass and impaired growth, including leaf symmetry loss and leaf deformities possibly related to contaminants, particularly heavy metals. Accordingly, we conclude that SS exhibited potential as a fertilizer at concentrations below 50%, but exerted a toxic effect on seedlings at higher concentrations.

Background: Inherited cardiomyopathy associates with a range of phenotypes, mediated by genetic and nongenetic factors. Noninherited cardiomyopathy also displays varying progression and outcomes. Expression of cardiomyopathy genes is under the regulatory control of promoters and enhancers, and human genetic variation in promoters and enhancers may contribute to this variability., Methods: We superimposed epigenomic profiling from hearts and cardiomyocytes, including promoter-capture chromatin conformation information, to identify enhancers for 2 cardiomyopathy genes, MYH7 and LMNA . Enhancer function was validated in human cardiomyocytes derived from induced pluripotent stem cells. We also conducted a genome-wide search to ascertain genomic variation in enhancers positioned to alter cardiac expression and correlated one of these variants to cardiomyopathy progression using biobank data., Results: Multiple enhancers were identified and validated for LMNA and MYH7 , including a key enhancer that regulates the switch from MYH6 expression to MYH7 expression. Deletion of this enhancer resulted in a dose-dependent increase in MYH6 and faster contractile rate in engineered heart tissues. We searched for genomic variation in enhancer sequences across the genome, with a focus on nucleotide changes that create or interrupt transcription factor binding sites. The sequence variant, rs875908, disrupts a T-Box Transcription Factor 5 binding motif and maps to an enhancer region 2 kilobases from the transcriptional start site of MYH7. Gene editing to remove the enhancer that harbors this variant markedly reduced MYH7 expression in human cardiomyocytes. Using biobank-derived data, rs875908 associated with longitudinal echocardiographic features of cardiomyopathy., Conclusions: Enhancers regulate cardiomyopathy gene expression, and genomic variation within these enhancer regions associates with cardiomyopathic progression over time. This integrated approach identified noncoding modifiers of cardiomyopathy and is applicable to other cardiac genes.

There is a life-long relationship between rhinovirus (RV) infection and the development and clinical manifestations of asthma. In this study we demonstrate that cultured primary bronchial epithelial cells from adults with asthma (n = 9) show different transcriptional and chromatin responses to RV infection compared to those without asthma (n = 9). Both the number and magnitude of transcriptional and chromatin responses to RV were muted in cells from asthma cases compared to controls. Pathway analysis of the transcriptionally responsive genes revealed enrichments of apoptotic pathways in controls but inflammatory pathways in asthma cases. Using promoter capture Hi-C we tethered regions of RV-responsive chromatin to RV-responsive genes and showed enrichment of these regions and genes at asthma GWAS loci. Taken together, our studies indicate a delayed or prolonged inflammatory state in cells from asthma cases and highlight genes that may contribute to genetic risk for asthma.

Background: The failing heart is characterized by changes in gene expression. However, the regulatory regions of the genome that drive these gene expression changes have not been well defined in human hearts., Methods: To define genome-wide enhancer and promoter use in heart failure, cap analysis of gene expression sequencing was applied to 3 healthy and 4 failed human hearts to identify promoter and enhancer regions used in left ventricles. Healthy hearts were derived from donors unused for transplantation and failed hearts were obtained as discarded tissue after transplantation., Results: Cap analysis of gene expression sequencing identified a combined potential for ≈23 000 promoters and ≈5000 enhancers active in human left ventricles. Of these, 17 000 promoters and 1800 enhancers had additional support for their regulatory function. Comparing promoter usage between healthy and failed hearts highlighted promoter shifts which altered aminoterminal protein sequences. Enhancer usage between healthy and failed hearts identified a majority of differentially used heart failure enhancers were intronic and primarily localized within the first intron, revealing this position as a common feature associated with tissue-specific gene expression changes in the heart., Conclusions: This data set defines the dynamic genomic regulatory landscape underlying heart failure and serves as an important resource for understanding genetic contributions to cardiac dysfunction. Additionally, regulatory changes contributing to heart failure are attractive therapeutic targets for controlling ventricular remodeling and clinical progression.

Recent revolutionary advances at the intersection of medicine, omics, data sciences, computing, epidemiology, and related technologies inspire us to ponder their impact on health. Their potential impact is particularly germane to the biology of pregnancy and perinatal medicine, where limited improvement in health outcomes for women and children has remained a global challenge. We assembled a group of experts to establish a Pregnancy Think Tank to discuss a broad spectrum of major gestational disorders and adverse pregnancy outcomes that affect maternal-infant lifelong health and should serve as targets for leveraging the many recent advances. This report reflects avenues for future effects that hold great potential in 3 major areas: developmental genomics, including the application of methodologies designed to bridge genotypes, physiology, and diseases, addressing vexing questions in early human development; gestational physiology, from immune tolerance to growth and the timing of parturition; and personalized and population medicine, focusing on amalgamating health record data and deep phenotypes to create broad knowledge that can be integrated into healthcare systems and drive discovery to address pregnancy-related disease and promote general health. We propose a series of questions reflecting development, systems biology, diseases, clinical approaches and tools, and population health, and a call for scientific action. Clearly, transdisciplinary science must advance and accelerate to address adverse pregnancy outcomes. Disciplines not traditionally involved in the reproductive sciences, such as computer science, engineering, mathematics, and pharmacology, should be engaged at the study design phase to optimize the information gathered and to identify and further evaluate potentially actionable therapeutic targets. Information sources should include noninvasive personalized sensors and monitors, alongside instructive "liquid biopsies" for noninvasive pregnancy assessment. Future research should also address the diversity of human cohorts in terms of geography, racial and ethnic distributions, and social and health disparities. Modern technologies, for both data-gathering and data-analyzing, make this possible at a scale that was previously unachievable. Finally, the psychosocial and economic environment in which pregnancy takes place must be considered to promote the health and wellness of communities worldwide., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Rationale: The heartbeat is organized by the cardiac conduction system (CCS), a specialized network of cardiomyocytes. Patterning of the CCS into atrial node versus ventricular conduction system (VCS) components with distinct physiology is essential for the normal heartbeat. Distinct node versus VCS physiology has been recognized for more than a century, but the molecular basis of this regional patterning is not well understood., Objective: To study the genetic and genomic mechanisms underlying node versus VCS distinction and investigate rhythm consequences of failed VCS patterning., Methods and Results: Using mouse genetics, we found that the balance between T-box transcriptional activator, Tbx5 , and T-box transcriptional repressor, Tbx3 , determined the molecular and functional output of VCS myocytes. Adult VCS-specific removal of Tbx5 or overexpression of Tbx3 re-patterned the fast VCS into slow, nodal-like cells based on molecular and functional criteria. In these cases, gene expression profiling showed diminished expression of genes required for VCS-specific fast conduction but maintenance of expression of genes required for nodal slow conduction physiology. Action potentials of Tbx5 -deficient VCS myocytes adopted nodal-specific characteristics, including increased action potential duration and cellular automaticity. Removal of Tbx5 in vivo precipitated inappropriate depolarizations in the atrioventricular (His)-bundle associated with lethal ventricular arrhythmias. TBX5 bound and directly activated cis -regulatory elements at fast conduction channel genes required for fast physiological characteristics of the VCS action potential, defining the identity of the adult VCS., Conclusions: The CCS is patterned entirely as a slow, nodal ground state, with a T-box dependent, physiologically dominant, fast conduction network driven specifically in the VCS. Disruption of the fast VCS gene regulatory network allowed nodal physiology to emerge, providing a plausible molecular mechanism for some lethal ventricular arrhythmias.

Disease-associated genetic variants that lie in non-coding regions found by genome-wide association studies are thought to alter the functionality of transcription regulatory elements and target gene expression. To uncover causal genetic variants, variant regulatory elements and their target genes, here we cross-reference human transcriptomic, epigenomic and chromatin conformation datasets. Of 104 genetic variant regions associated with atrial fibrillation candidate target genes are prioritized. We optimize EMERGE enhancer prediction and use accessible chromatin profiles of human atrial cardiomyocytes to more accurately predict cardiac regulatory elements and identify hundreds of sub-threshold variants that co-localize with regulatory elements. Removal of mouse homologues of atrial fibrillation-associated regions in vivo uncovers a distal regulatory region involved in Gja1 (Cx43) expression. Our analyses provide a shortlist of genes likely affected by atrial fibrillation-associated variants and provide variant regulatory elements in each region that link genetic variation and target gene regulation, helping to focus future investigations.

Rationale: Mutations in the transcription factor TBX20 (T-box 20) are associated with congenital heart disease. Germline ablation of Tbx20 results in abnormal heart development and embryonic lethality by embryonic day 9.5. Because Tbx20 is expressed in multiple cell lineages required for myocardial development, including pharyngeal endoderm, cardiogenic mesoderm, endocardium, and myocardium, the cell type-specific requirement for TBX20 in early myocardial development remains to be explored., Objective: Here, we investigated roles of TBX20 in midgestation cardiomyocytes for heart development., Methods and Results: Ablation of Tbx20 from developing cardiomyocytes using a doxycycline inducible cTnTCre transgene led to embryonic lethality. The circumference of developing ventricular and atrial chambers, and in particular that of prospective left atrium, was significantly reduced in Tbx20 conditional knockout mutants. Cell cycle analysis demonstrated reduced proliferation of Tbx20 mutant cardiomyocytes and their arrest at the G1-S phase transition. Genome-wide transcriptome analysis of mutant cardiomyocytes revealed differential expression of multiple genes critical for cell cycle regulation. Moreover, atrial and ventricular gene programs seemed to be aberrantly regulated. Putative direct TBX20 targets were identified using TBX20 ChIP-Seq (chromatin immunoprecipitation with high throughput sequencing) from embryonic heart and included key cell cycle genes and atrial and ventricular specific genes. Notably, TBX20 bound a conserved enhancer for a gene key to atrial development and identity, COUP-TFII/Nr2f2 (chicken ovalbumin upstream promoter transcription factor 2/nuclear receptor subfamily 2, group F, member 2). This enhancer interacted with the NR2F2 promoter in human cardiomyocytes and conferred atrial specific gene expression in a transgenic mouse in a TBX20-dependent manner., Conclusions: Myocardial TBX20 directly regulates a subset of genes required for fetal cardiomyocyte proliferation, including those required for the G1-S transition. TBX20 also directly downregulates progenitor-specific genes and, in addition to regulating genes that specify chamber versus nonchamber myocardium, directly activates genes required for establishment or maintenance of atrial and ventricular identity. TBX20 plays a previously unappreciated key role in atrial development through direct regulation of an evolutionarily conserved COUPT-FII enhancer.

The epigenome provides a substrate through which environmental exposures can exert their effects on gene expression and disease risk, but the relative importance of epigenetic variation on human disease onset and progression is poorly characterized. Asthma is a heterogeneous disease of the airways, for which both onset and clinical course result from interactions between host genotype and environmental exposures, yet little is known about the molecular mechanisms for these interactions. We assessed genome-wide DNA methylation using the Infinium Human Methylation 450K Bead Chip and characterized the transcriptome by RNA sequencing in primary airway epithelial cells from 74 asthmatic and 41 nonasthmatic adults. Asthma status was based on doctor's diagnosis and current medication use. Genotyping was performed using various Illumina platforms. Our study revealed a regulatory locus on chromosome 17q12-21 associated with asthma risk and epigenetic signatures of specific asthma endotypes and molecular networks. Overall, these data support a central role for DNA methylation in lung cells, which promotes distinct molecular pathways of asthma pathogenesis and modulates the effects of genetic variation on disease risk and clinical heterogeneity., Competing Interests: J. Solway has been a scientific advisor for and has a financial interest in PulmOne Advanced Medical Devices Ltd., Israel, and received reimbursement for expenses. He served on the Respiratory Therapy Clinical Advisory Board for Hollister Inc., and for this received honoraria and was reimbursed for travel and meal expenses incurred during meetings. He has received a research grant from AstraZeneca Inc., from 2006 to 2014, that was administered through the University of Chicago. He has multiple patents concerning a smooth muscle gene promoter and one pending concerning a method to determine respiratory physiological parameters (6090618; 6114311; 6284743; 6291211; 6297221; 6331527; 7169764). He has consulted for Novartis Institute for Biomedical Research, for which he received an honorarium and travel reimbursement. He was a member of the scientific advisory board for Cytokinetics Inc., for which he received honoraria and travel reimbursement.

Mutations in the T-box transcription factor TBX20 are associated with multiple forms of congenital heart defects, including cardiac septal abnormalities, but our understanding of the contributions of endocardial TBX20 to heart development remains incomplete. Here, we investigated how TBX20 interacts with endocardial gene networks to drive the mesenchymal and myocardial movements that are essential for outflow tract and atrioventricular septation. Selective ablation of Tbx20 in murine endocardial lineages reduced the expression of extracellular matrix and cell migration genes that are critical for septation. Using the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), we identified accessible chromatin within endocardial lineages and intersected these data with TBX20 ChIP-seq and chromatin loop maps to determine that TBX20 binds a conserved long-range enhancer to regulate versican (Vcan) expression. We also observed reduced Vcan expression in Tbx20-deficient mice, supporting a direct role for TBX20 in Vcan regulation. Further, we show that the Vcan enhancer drove reporter gene expression in endocardial lineages in a TBX20-binding site-dependent manner. This work illuminates gene networks that interact with TBX20 to orchestrate cardiac septation and provides insight into the chromatin landscape of endocardial lineages during septation.

Increasing evidence in the last years indicates that the vast amount of regulatory information contained in mammalian genomes is organized in precise 3D chromatin structures. However, the impact of this spatial chromatin organization on gene expression and its degree of evolutionary conservation is still poorly understood. The Six homeobox genes are essential developmental regulators organized in gene clusters conserved during evolution. Here, we reveal that the Six clusters share a deeply evolutionarily conserved 3D chromatin organization that predates the Cambrian explosion. This chromatin architecture generates two largely independent regulatory landscapes (RLs) contained in two adjacent topological associating domains (TADs). By disrupting the conserved TAD border in one of the zebrafish Six clusters, we demonstrate that this border is critical for preventing competition between promoters and enhancers located in separated RLs, thereby generating different expression patterns in genes located in close genomic proximity. Moreover, evolutionary comparison of Six-associated TAD borders reveals the presence of CCCTC-binding factor (CTCF) sites with diverging orientations in all studied deuterostomes. Genome-wide examination of mammalian HiC data reveals that this conserved CTCF configuration is a general signature of TAD borders, underscoring that common organizational principles underlie TAD compartmentalization in deuterostome evolution.

Transcriptional mediators of cell stress pathways, including HIF1α, ATF4, and p53, are key to normal development and play critical roles in disease, including ischemia and cancer. Despite their importance, mechanisms by which pathways mediated by these transcription factors interact with one another are not fully understood. In addressing the controversial role of HIF1α in cardiomyocytes (CMs) during heart development, we discovered a mid-gestational requirement for HIF1α for proliferation of hypoxic CMs, involving metabolic switching and a complex interplay among HIF1α, ATF4, and p53. Loss of HIF1α resulted in activation of ATF4 and p53, the latter inhibiting CM proliferation. Bioinformatic and biochemical analyses revealed unexpected mechanisms by which HIF1α intersects with ATF4 and p53 pathways. Our results highlight previously undescribed roles of HIF1α and interactions among major cell stress pathways that could be targeted to enhance proliferation of CMs in ischemia and may have relevance to other diseases, including cancer., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Organisms with targeted genomic modifications are efficiently produced by gene editing in embryos using CRISPR/Cas9 RNA-guided DNA endonuclease. Here, to facilitate germline editing in rats, we used CRISPR/Cas9 to catalyze targeted genomic mutations in rat spermatogonial stem cell cultures. CRISPR/Cas9-modified spermatogonia regenerated spermatogenesis and displayed long-term sperm-forming potential following transplantation into rat testes. Targeted germline mutations in Epsti1 and Erbb3 were vertically transmitted from recipients to exclusively generate "pure," non-mosaic mutant progeny. Epsti1 mutant rats were produced with or without genetic selection of donor spermatogonia. Monoclonal enrichment of Erbb3 null germlines unmasked recessive spermatogenesis defects in culture that were buffered in recipients, yielding mutant progeny isogenic at targeted alleles. Thus, spermatogonial gene editing with CRISPR/Cas9 provided a platform for generating targeted germline mutations in rats and for studying spermatogenesis., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Non-coding variation within TCF7L2 remains the strongest genetic determinant of type 2 diabetes risk in humans. A considerable effort has been placed in understanding the functional roles of TCF7L2 in pancreatic beta cells, despite evidence of TCF7L2 expression in various peripheral tissues important in glucose homeostasis. Here, we use a humanized mouse model overexpressing Tcf7l2, resulting in glucose intolerance, to infer the contribution of Tcf7l2 overexpression in beta cells and in other tissues to the metabolic phenotypes displayed by these mice. Restoring Tcf7l2 expression specifically in beta cells to endogenous levels, in face of its overexpression elsewhere, results in impaired insulin secretion, reduced beta cell number and islet area, corroborating data obtained in humans showing similar phenotypes as a result of manipulations leading to Tcf7l2 loss of function. Interestingly, the persistent overexpression of Tcf7l2 in non-pancreatic tissues results in a significant worsening in glucose tolerance in vivo, indicating that Tcf7l2 overexpression in beta cells does not account for the glucose intolerance in the Tcf7l2 overexpression mouse model. Collectively, these data posit that Tcf7l2 plays key roles in glucose metabolism through actions beyond pancreatic beta cells, and further points to functionally opposing cell-type specific effects for Tcf7l2 on the maintenance of balanced glucose metabolism, thereby urging a careful examination of its role in non-pancreatic tissues as well as its composite metabolic effects across distinct tissues. Uncovering these roles may lead to new therapeutic targets for type 2 diabetes., (© The Author 2014. Published by Oxford University Press.)

Sleep disorders are highly prevalent during late pregnancy and can impose adverse effects, such as preeclampsia and diabetes. However, the consequences of sleep fragmentation (SF) on offspring metabolism and epigenomic signatures are unclear. We report that physical activity during early life, but not later, reversed the increased body weight, altered glucose and lipid homeostasis, and increased visceral adipose tissue in offspring of mice subjected to gestational SF (SFo). The reversibility of this phenotype may reflect epigenetic mechanisms induced by SF during gestation. Accordingly, we found that the metabolic master switch Foxo1 was epigenetically misregulated in SFo livers in a temporally regulated fashion. Temporal Foxo1 analysis and its gluconeogenetic targets revealed that the epigenetic abnormalities of Foxo1 precede the metabolic syndrome phenotype. Importantly, regular physical activity early, but not later in life, reversed Foxo1 epigenetic misregulation and altered the metabolic phenotype in gestationally SF-exposed offspring. Thus, we have identified a restricted postnatal period during which lifestyle interventions may reverse the Foxo1 epigenetically mediated risk for metabolic dysfunction later in the life, as induced by gestational sleep disorders., (Copyright © 2015 the American Physiological Society.)

Mixed lineage leukemia (MLL) fusion proteins directly activate the expression of key downstream genes such as MEIS1, HOXA9 to drive an aggressive form of human leukemia. However, it is still poorly understood what additional transcriptional regulators, independent of the MLL fusion pathway, contribute to the development of MLL leukemia. Here we show that the transcription factor PU.1 is essential for MLL leukemia and is required for the growth of MLL leukemic cells via the promotion of cell-cycle progression and inhibition of apoptosis. Importantly, PU.1 expression is not under the control of MLL fusion proteins. We further identified a PU.1-governed 15-gene signature, which contains key regulators in the MEIS-HOX program (MEIS1, PBX3, FLT3, and c-KIT). PU.1 directly binds to the genomic loci of its target genes in vivo, and is required to maintain active expression of those genes in both normal hematopoietic stem and progenitor cells and in MLL leukemia. Finally, the clinical significance of the identified PU.1 signature was indicated by its ability to predict survival in acute myelogenous leukemia patients. Together, our findings demonstrate that PU.1 contributes to the development of MLL leukemia, partially via crosstalk with the MEIS/HOX pathway.

Variants in SCN10A, which encodes a voltage-gated sodium channel, are associated with alterations of cardiac conduction parameters and the cardiac rhythm disorder Brugada syndrome; however, it is unclear how SCN10A variants promote dysfunctional cardiac conduction. Here we showed by high-resolution 4C-seq analysis of the Scn10a-Scn5a locus in murine heart tissue that a cardiac enhancer located in Scn10a, encompassing SCN10A functional variant rs6801957, interacts with the promoter of Scn5a, a sodium channel-encoding gene that is critical for cardiac conduction. We observed that SCN5A transcript levels were several orders of magnitude higher than SCN10A transcript levels in both adult human and mouse heart tissue. Analysis of BAC transgenic mouse strains harboring an engineered deletion of the enhancer within Scn10a revealed that the enhancer was essential for Scn5a expression in cardiac tissue. Furthermore, the common SCN10A variant rs6801957 modulated Scn5a expression in the heart. In humans, the SCN10A variant rs6801957, which correlated with slowed conduction, was associated with reduced SCN5A expression. These observations establish a genomic mechanism for how a common genetic variation at SCN10A influences cardiac physiology and predisposes to arrhythmia.

Aberrant activation of the three-amino-acid-loop extension homeobox gene MEIS1 shortens the latency and accelerates the onset and progression of acute leukemia, yet the molecular mechanism underlying persistent activation of the MEIS1 gene in leukemia remains poorly understood. Here we used a combined comparative genomics analysis and an in vivo transgenic zebrafish assay to identify six regulatory DNA elements that are able to direct green fluorescent protein expression in a spatiotemporal manner during zebrafish embryonic hematopoiesis. Analysis of chromatin characteristics and regulatory signatures suggests that many of these predicted elements are potential enhancers in mammalian hematopoiesis. Strikingly, one of the enhancer elements (E9) is a frequent integration site in retroviral-induced mouse acute leukemia. The genomic region corresponding to enhancer E9 is differentially marked by H3K4 monomethylation and H3K27 acetylation, hallmarks of active enhancers, in multiple leukemia cell lines. Decreased enrichment of these histone marks is associated with downregulation of MEIS1 expression during hematopoietic differentiation. Further, MEIS1/HOXA9 transactivate this enhancer via a conserved binding motif in vitro, and participate in an autoregulatory loop that modulates MEIS1 expression in vivo. Our results suggest that an intronic enhancer regulates the expression of MEIS1 in hematopoiesis and contributes to its aberrant expression in acute leukemia.

The complex expression patterns observed for many genes are often regulated by distal transcription enhancers. Changes in the nucleotide sequences of enhancers may therefore lead to changes in gene expression, representing a central mechanism by which organisms evolve. With the development of the experimental technique of chromatin immunoprecipitation (ChIP), in which discrete regions of the genome bound by specific proteins can be identified, it is now possible to identify transcription factor binding events (putative cis-regulatory elements) in entire genomes. Comparing protein-DNA binding maps allows us, for the first time, to attempt to identify regulatory differences and infer global patterns of change in gene expression across species. Here, we review studies that used genome-wide ChIP to study the evolution of enhancers. The trend is one of high divergence of cis-regulatory elements between species, possibly compensated by extensive creation and loss of regulatory elements and rewiring of their target genes. We speculate on the meaning of the differences observed and discuss that although ChIP experiments identify the biochemical event of protein-DNA interaction, it cannot determine whether the event results in a biological function, and therefore more studies are required to establish the effect of divergence of binding events on species-specific gene expression.

Although long known and the most prevalent movement disorder, pathophysiology of essential tremor (ET) remains controversial. The most accepted hypothesis is that it is caused by a dysfunction of the olivocerebellar system. Vilela Filho et al. [2001; Stereotact Funct Neurosurg 77:149-150], however, reported a patient with unilateral hand ET that was completely relieved after a stroke restricted to the contralateral posterior putamen and suggested that ET could be the clinical manifestation of posterior putamen hyperactivity. The present study was designed to evaluate this hypothesis in the most often used model of ET, harmaline-induced tremor in rats. Fifty-four male Wistar rats were randomly distributed into three groups: experimental (EG), surgical control (SCG), and pharmacological control (PCG) groups. EG animals underwent stereotactic unilateral posterior striatotomy. SCG rats underwent sham lesion at the same target. PCG served exclusively as controls for harmaline effects. All animals received, postoperatively, intraperitoneal harmaline, and the induced tremor was video-recorded for later evaluation by a blind observer. Thirteen animals were excluded from the study. Limb tremor was reduced ipsilaterally to the operation in 20 of 21 rats of EG and in two of nine of SCG, being asymmetric in one of 10 of PCG rats. Comparisons between EG × SCG and EG × PCG were statistically significant, but not between SCG × PCG. Limb tremor reduction was greater in anterior than in posterior paws. Lateral lesions yielded better results than medial lesions. These results suggest that the posterior striatum is involved with harmaline-induced tremor in rats and support the hypothesis presented., (Copyright © 2013 Wiley Periodicals, Inc.)

N(6)-methyladenosine is a prevalent internal modification in messenger RNA and non-coding RNA affecting various cellular pathways. Here we report the discovery of two additional modifications, N(6)-hydroxymethyladenosine (hm(6)A) and N(6)-formyladenosine (f(6)A), in mammalian messenger RNA. We show that Fe(II)- and α-ketoglutarate-dependent fat mass and obesity-associated (FTO) protein oxidize N(6)-methyladenosine to generate N(6)-hydroxymethyladenosine as an intermediate modification, and N(6)-formyladenosine as a further oxidized product. N(6)-hydroxymethyladenosine and N(6)-formyladenosine have half-life times of ~3 h in aqueous solution under physiological relevant conditions, and are present in isolated messenger RNA from human cells as well as mouse tissues. These previously unknown modifications derived from the prevalent N(6)-methyladenosine in messenger RNA, formed through oxidative RNA demethylation, may dynamically modulate RNA-protein interactions to affect gene expression regulation.

Aims/hypothesis: Recent functional characterisations of genome-wide association study (GWAS) loci suggest that cis-regulatory variation may be a common paradigm for complex disease susceptibility. Several studies point to a similar mechanism at the transcription factor 7-like 2 (TCF7L2) GWAS locus for type 2 diabetes. To address this possibility, we carried out an in vitro scan of this diabetes-associated locus to fine-map cis-regulatory sequences within this genomic interval., Methods: A systematic cell-based enhancer strategy was employed to interrogate all sequences within the 92 kb type-2-diabetes-association interval for cis-regulatory activity in a panel of cell lines (HCT-116, Neuro-2a, C2C12, U2OS, MIN6 and HepG2). We further evaluated chromatin state at a subset of these regions in HCT-116 and U2OS cells and examined allelic-specific enhancer properties at the type-2-diabetes-associated single nucleotide polymorphism (SNP) rs7903146., Results: In total, we assigned cis-regulatory activity to approximately 30% (9/28) of constructs tested. Notably, a subset of enhancers was active across multiple cell lines and overlapped with key epigenetic markers suggestive of cis-regulatory sequences. We further replicated the allelic-specific properties for SNP rs7903146 in pancreatic beta cells and additionally demonstrate identical allelic-specific enhancer effects in other cell lines., Conclusions: These results provide a detailed map of cis-regulatory elements within the TCF7L2 GWAS locus and support the hypothesis of cis-regulatory variation leading to type 2 diabetes predisposition. The detection of allelic-specific effects for SNP rs7903146 in multiple cell lines further alludes to the likelihood of a peripheral defect in disease aetiology.

Recent studies have identified the genetic underpinnings of a growing number of diseases through targeted exome sequencing. However, this strategy ignores the large component of the genome that does not code for proteins, but is nonetheless biologically functional. To address the possible involvement of regulatory variation in congenital heart diseases (CHDs), we searched for regulatory mutations impacting the activity of TBX5, a dosage-dependent transcription factor with well-defined roles in the heart and limb development that has been associated with the Holt-Oram syndrome (heart-hand syndrome), a condition that affects 1/100 000 newborns. Using a combination of genomics, bioinformatics and mouse genetic engineering, we scanned ∼700 kb of the TBX5 locus in search of cis-regulatory elements. We uncovered three enhancers that collectively recapitulate the endogenous expression pattern of TBX5 in the developing heart. We re-sequenced these enhancer elements in a cohort of non-syndromic patients with isolated atrial and/or ventricular septal defects, the predominant cardiac defects of the Holt-Oram syndrome, and identified a patient with a homozygous mutation in an enhancer ∼90 kb downstream of TBX5. Notably, we demonstrate that this single-base-pair mutation abrogates the ability of the enhancer to drive expression within the heart in vivo using both mouse and zebrafish transgenic models. Given the population-wide frequency of this variant, we estimate that 1/100 000 individuals would be homozygous for this variant, highlighting that a significant number of CHD associated with TBX5 dysfunction might arise from non-coding mutations in TBX5 heart enhancers, effectively decoupling the heart and hand phenotypes of the Holt-Oram syndrome.

Cardiac conduction system (CCS) disease, which results in disrupted conduction and impaired cardiac rhythm, is common with significant morbidity and mortality. Current treatment options are limited, and rational efforts to develop cell-based and regenerative therapies require knowledge of the molecular networks that establish and maintain CCS function. Recent genome-wide association studies (GWAS) have identified numerous loci associated with adult human CCS function, including TBX5 and SCN5A. We hypothesized that TBX5, a critical developmental transcription factor, regulates transcriptional networks required for mature CCS function. We found that deletion of Tbx5 from the mature murine ventricular conduction system (VCS), including the AV bundle and bundle branches, resulted in severe VCS functional consequences, including loss of fast conduction, arrhythmias, and sudden death. Ventricular contractile function and the VCS fate map remained unchanged in VCS-specific Tbx5 knockouts. However, key mediators of fast conduction, including Nav1.5, which is encoded by Scn5a, and connexin 40 (Cx40), demonstrated Tbx5-dependent expression in the VCS. We identified a TBX5-responsive enhancer downstream of Scn5a sufficient to drive VCS expression in vivo, dependent on canonical T-box binding sites. Our results establish a direct molecular link between Tbx5 and Scn5a and elucidate a hierarchy between human GWAS loci that affects function of the mature VCS, establishing a paradigm for understanding the molecular pathology of CCS disease.

Glyoxalase 1 (Glo1) expression has previously been associated with anxiety in mice; however, its role in anxiety is controversial, and the underlying mechanism is unknown. Here, we demonstrate that GLO1 increases anxiety by reducing levels of methylglyoxal (MG), a GABAA receptor agonist. Mice overexpressing Glo1 on a Tg bacterial artificial chromosome displayed increased anxiety-like behavior and reduced brain MG concentrations. Treatment with low doses of MG reduced anxiety-like behavior, while higher doses caused locomotor depression, ataxia, and hypothermia, which are characteristic effects of GABAA receptor activation. Consistent with these data, we found that physiological concentrations of MG selectively activated GABAA receptors in primary neurons. These data indicate that GLO1 increases anxiety by reducing levels of MG, thereby decreasing GABAA receptor activation. More broadly, our findings potentially link metabolic state, neuronal inhibitory tone, and behavior. Finally, we demonstrated that pharmacological inhibition of GLO1 reduced anxiety, suggesting that GLO1 is a possible target for the treatment of anxiety disorders.

The ongoing requirement in adult heart for transcription factors with key roles in cardiac development is not well understood. We recently demonstrated that TBX20, a transcriptional regulator required for cardiac development, has key roles in the maintenance of functional and structural phenotypes in adult mouse heart. Conditional ablation of Tbx20 in adult cardiomyocytes leads to a rapid onset and progression of heart failure, with prominent conduction and contractility phenotypes that lead to death. Here we describe a more comprehensive molecular characterization of the functions of TBX20 in adult mouse heart. Coupling genome-wide chromatin immunoprecipitation and transcriptome analyses (RNA-Seq), we identified a subset of genes that change expression in Tbx20 adult cardiomyocyte-specific knockout hearts which are direct downstream targets of TBX20. This analysis revealed a dual role for TBX20 as both a transcriptional activator and a repressor, and that each of these functions regulates genes with very specialized and distinct molecular roles. We also show how TBX20 binds to its targets genome-wide in a context-dependent manner, using various cohorts of co-factors to either promote or repress distinct genetic programs within adult heart. Our integrative approach has uncovered several novel aspects of TBX20 and T-box protein function within adult heart. Sequencing data accession number (http://www.ncbi.nlm.nih.gov/geo): GSE30943.

To study the evolution of recombination rates in apes, we developed methodology to construct a fine-scale genetic map from high-throughput sequence data from 10 Western chimpanzees, Pan troglodytes verus. Compared to the human genetic map, broad-scale recombination rates tend to be conserved, but with exceptions, particularly in regions of chromosomal rearrangements and around the site of ancestral fusion in human chromosome 2. At fine scales, chimpanzee recombination is dominated by hotspots, which show no overlap with those of humans even though rates are similarly elevated around CpG islands and decreased within genes. The hotspot-specifying protein PRDM9 shows extensive variation among Western chimpanzees, and there is little evidence that any sequence motifs are enriched in hotspots. The contrasting locations of hotspots provide a natural experiment, which demonstrates the impact of recombination on base composition.

Distal transcription enhancers are cis-regulatory elements that promote gene expression, enabling spatiotemporal control of genetic programs such as those required in metazoan developmental processes. Because of their importance, their disruption can lead to disease.

Genome-wide association studies (GWAS) have repeatedly shown an association between non-coding variants in the TCF7L2 locus and risk for type 2 diabetes (T2D), implicating a role for cis-regulatory variation within this locus in disease etiology. Supporting this hypothesis, we previously localized complex regulatory activity to the TCF7L2 T2D-associated interval using an in vivo bacterial artificial chromosome (BAC) enhancer-trapping reporter strategy. To follow-up on this broad initial survey of the TCF7L2 regulatory landscape, we performed a fine-mapping enhancer scan using in vivo mouse transgenic reporter assays. We functionally interrogated approximately 50% of the sequences within the T2D-associated interval, utilizing sequence conservation within this 92-kb interval to determine the regulatory potential of all evolutionary conserved sequences that exhibited conservation to the non-eutherian mammal opossum. Included in this study was a detailed functional interrogation of sequences spanning both protective and risk alleles of single nucleotide polymorphism (SNP) rs7903146, which has exhibited allele-specific enhancer function in pancreatic beta cells. Using these assays, we identified nine segments regulating various aspects of the TCF7L2 expression profile and that constitute nearly 70% of the sequences tested. These results highlight the regulatory complexity of this interval and support the notion that a TCF7L2 cis-regulatory disruption leads to T2D predisposition.

Human mutations in or variants of TBX20 are associated with congenital heart disease, cardiomyopathy, and arrhythmias. To investigate whether cardiac disease in patients with these conditions results from an embryonic or ongoing requirement for Tbx20 in myocardium, we ablated Tbx20 specifically in adult cardiomyocytes in mice. This ablation resulted in the onset of severe cardiomyopathy accompanied by arrhythmias, with death ensuing within 1 to 2 weeks of Tbx20 ablation. Accounting for this dramatic phenotype, we identified molecular signatures that posit Tbx20 as a central integrator of a genetic program that maintains cardiomyocyte function in the adult heart. Expression of a number of genes encoding critical transcription factors, ion channels, and cytoskeletal/myofibrillar proteins was downregulated consequent to loss of Tbx20. Genome-wide ChIP analysis of Tbx20-binding regions in the adult heart revealed that many of these genes were direct downstream targets of Tbx20 and uncovered a previously undescribed DNA-binding site for Tbx20. Bioinformatics and in vivo functional analyses revealed a cohort of transcription factors that, working with Tbx20, integrated multiple environmental signals to maintain ion channel gene expression in the adult heart. Our data provide insight into the mechanisms by which mutations in TBX20 cause adult heart disease in humans.

Genome-wide association studies (GWAS) have consistently implicated noncoding variation within the TCF7L2 locus with type 2 diabetes (T2D) risk. While this locus represents the strongest genetic determinant for T2D risk in humans, it remains unclear how these noncoding variants affect disease etiology. To test the hypothesis that the T2D-associated interval harbors cis-regulatory elements controlling TCF7L2 expression, we conducted in vivo transgenic reporter assays to characterize the TCF7L2 regulatory landscape. We found that the 92-kb genomic interval associated with T2D harbors long-range enhancers regulating various aspects of the spatial-temporal expression patterns of TCF7L2, including expression in tissues involved in the control of glucose homeostasis. By selectively deleting this interval, we establish a critical role for these enhancers in robust TCF7L2 expression. To further determine whether variation in Tcf7l2 expression may lead to diabetes, we developed a Tcf7l2 copy-number allelic series in mice. We show that a null Tcf7l2 allele leads, in a dose-dependent manner, to lower glycemic profiles. Tcf7l2 null mice also display enhanced glucose tolerance coupled to significantly lowered insulin levels, suggesting that these mice are protected against T2D. Confirming these observations, transgenic mice harboring multiple Tcf7l2 copies and overexpressing this gene display reciprocal phenotypes, including glucose intolerance. These results directly demonstrate that Tcf7l2 plays a role in regulating glucose tolerance, suggesting that overexpression of this gene is associated with increased risk of T2D. These data highlight the role of enhancer elements as mediators of T2D risk in humans, strengthening the evidence that variation in cis-regulatory elements may be a paradigm for genetic predispositions to common disease.

The evolutionary transition of the fins of fish into tetrapod limbs involved genetic changes to developmental systems that resulted in novel skeletal patterns and functions. Approaches to understanding this issue have entailed the search for antecedents of limb structure in fossils, genes, and embryos. Comparative genetic analyses have produced ambiguous results: although studies of posterior Hox genes from homology group 13 (Hoxa-13 and Hoxd-13) reveal similarities in gene expression between the distal segments of fins and limbs, this functional homology has not been supported by genomic comparisons of the activity of their cis-regulatory elements, namely the Hoxd Global Control Region. Here, we show that cis-regulatory elements driving Hoxd gene expression in distal limbs are present in fish. Using an interspecies transgenesis approach, we find functional conservation between gnathostome Hoxd enhancers, demonstrating that orthologous sequences from tetrapods, zebrafish and skate can drive reporter gene expression in mouse limbs and zebrafish fins. Our results support the notion that some of the novelties associated with tetrapod limbs arose by modification of deeply conserved cis- and trans-acting mechanisms of Hox regulation in gnathostomes.

Plasticity of gene regulatory encryption can permit DNA sequence divergence without loss of function. Functional information is preserved through conservation of the composition of transcription factor binding sites (TFBS) in a regulatory element. We have developed a method that can accurately identify pairs of functional noncoding orthologs at evolutionarily diverged loci by searching for conserved TFBS arrangements. With an estimated 5% false-positive rate (FPR) in approximately 3000 human and zebrafish syntenic loci, we detected approximately 300 pairs of diverged elements that are likely to share common ancestry and have similar regulatory activity. By analyzing a pool of experimentally validated human enhancers, we demonstrated that 7/8 (88%) of their predicted functional orthologs retained in vivo regulatory control. Moreover, in 5/7 (71%) of assayed enhancer pairs, we observed concordant expression patterns. We argue that TFBS composition is often necessary to retain and sufficient to predict regulatory function in the absence of overt sequence conservation, revealing an entire class of functionally conserved, evolutionarily diverged regulatory elements that we term "covert."

Proper function of an organized Cardiac Conduction System (CCS) is vital to the survival of metazoans ranging from fly to man. The routine use of non-invasive electrocardiogram measures in the diagnosis and monitoring of cardiovascular health has established a trove of reliable CCS functional data in both normal and diseased cardiac states. Recent combination of echocardiogram (ECG) data with genome-wide association studies has identified genomic regions implicated in ECG variability which impact CCS function. In this study, we review the substantial recent progress in this area, highlighting the identification of novel loci, confirming the importance of previously implicated loci in CCS function, and exploring potential links between genes with important roles in developmental processes and variation in function of the CCS., (Copyright © 2011 Wiley-Liss, Inc.)