Bulk Segregant Analysis Using Single Nucleotide Polymorphism Microarrays

Bulk segregant analysis (BSA) using microarrays, and extreme array mapping (XAM) have recently been used to rapidly identify genomic regions associated with phenotypes in multiple species. These experiments, however, require the identification of single feature polymorphisms (SFP) between the cross parents for each new combination of genotypes, which raises the cost of experiments. The availability of the genomic polymorphism data in Arabidopsis thaliana, coupled with the efficient designs of Single Nucleotide Polymorphism (SNP) genotyping arrays removes the requirement for SFP detection and lowers the per array cost, thereby lowering the overall cost per experiment. To demonstrate that these approaches would be functional on SNP arrays and determine confidence intervals, we analyzed hybridizations of natural accessions to the Arabidopsis ATSNPTILE array and simulated BSA or XAM given a variety of gene models, populations, and bulk selection parameters. Our results show a striking degree of correlation between the genotyping output of both methods, which suggests that the benefit of SFP genotyping in context of BSA can be had with the cheaper, more efficient SNP arrays. As a final proof of concept, we hybridized the DNA from bulks of an F2 mapping population of a Sulfur and Selenium ionomics mutant to both the Arabidopsis ATTILE1R and ATSNPTILE arrays, which produced almost identical results. We have produced R scripts that prompt the user for the required parameters and perform the BSA analysis using the ATSNPTILE1 array and have provided them as supplemental data files

FTO influences adipogenesis by regulating mitotic clonal expansion

The fat mass and obesity-associated (FTO) gene plays a pivotal role in regulating body weight and fat mass; however, the underlying mechanisms are poorly understood. Here we show that primary adipocytes and mouse embryonic fibroblasts (MEFs) derived from FTO overexpression (FTO-4) mice exhibit increased potential for adipogenic differentiation, while MEFs derived from FTO knockout (FTO-KO) mice show reduced adipogenesis. As predicted from these findings, fat pads from FTO-4 mice fed a high-fat diet show more numerous adipocytes. FTO influences adipogenesis by regulating events early in adipogenesis, during the process of mitotic clonal expansion. The effect of FTO on adipogenesis appears to be mediated via enhanced expression of the pro-adipogenic short isoform of RUNX1T1, which enhanced adipocyte proliferation, and is increased in FTO-4 MEFs and reduced in FTO-KO MEFs. Our findings provide novel mechanistic insight into how upregulation of FTO leads to obesity

Integrative analysis of omics summary data reveals putative mechanisms underlying complex traits

The identification of genes and regulatory elements underlying the associations discovered by GWAS is essential to understanding the aetiology of complex traits (including diseases). Here, we demonstrate an analytical paradigm of prioritizing genes and regulatory elements at GWAS loci for follow-up functional studies. We perform an integrative analysis that uses summary-level SNP data from multi-omics studies to detect DNA methylation (DNAm) sites associated with gene expression and phenotype through shared genetic effects (i.e., pleiotropy). We identify pleiotropic associations between 7858 DNAm sites and 2733 genes. These DNAm sites are enriched in enhancers and promoters, and >40% of them are mapped to distal genes. Further pleiotropic association analyses, which link both the methylome and transcriptome to 12 complex traits, identify 149 DNAm sites and 66 genes, indicating a plausible mechanism whereby the effect of a genetic variant on phenotype is mediated by genetic regulation of transcription through DNAm

Single-Cell Expression Profiling Reveals a Dynamic State of Cardiac Precursor Cells in the Early Mouse Embryo

In the early vertebrate embryo, cardiac progenitor/precursor cells (CPs) give rise to cardiac structures. Better understanding their biological character is critical to understand the heart development and to apply CPs for the clinical arena. However, our knowledge remains incomplete. With the use of single-cell expression profiling, we have now revealed rapid and dynamic changes in gene expression profiles of the embryonic CPs during the early phase after their segregation from the cardiac mesoderm. Progressively, the nascent mesodermal gene Mesp1 terminated, and Nkx2-5+/Tbx5+ population rapidly replaced the Tbx5low+ population as the expression of the cardiac genes Tbx5 and Nkx2-5 increased. At the Early Headfold stage, Tbx5-expressing CPs gradually showed a unique molecular signature with signs of cardiomyocyte differentiation. Lineage-tracing revealed a developmentally distinct characteristic of this population. They underwent progressive differentiation only towards the cardiomyocyte lineage corresponding to the first heart field rather than being maintained as a progenitor pool. More importantly, Tbx5 likely plays an important role in a transcriptional network to regulate the distinct character of the FHF via a positive feedback loop to activate the robust expression of Tbx5 in CPs. These data expands our knowledge on the behavior of CPs during the early phase of cardiac development, subsequently providing a platform for further study

Anaerobic oxidation of methane associated with sulfate reduction in a natural freshwater gas source

The occurrence of anaerobic oxidation of methane (AOM) and trace methane oxidation (TMO) was investigated in a freshwater natural gas source. Sediment samples were taken and analyzed for potential electron acceptors coupled to AOM. Long-term incubations with 13C-labeled CH4 (13CH4) and different electron acceptors showed that both AOM and TMO occurred. In most conditions, 13C-labeled CO2 (13CO2) simultaneously increased with methane formation, which is typical for TMO. In the presence of nitrate, neither methane formation nor methane oxidation occurred. Net AOM was measured only with sulfate as electron acceptor. Here, sulfide production occurred simultaneously with 13CO2 production and no methanogenesis occurred, excluding TMO as a possible source for 13CO2 production from 13CH4. Archaeal 16S rRNA gene analysis showed the highest presence of ANME-2a/b (ANaerobic MEthane oxidizing archaea) and AAA (AOM Associated Archaea) sequences in the incubations with methane and sulfate as compared with only methane addition. Higher abundance of ANME-2a/b in incubations with methane and sulfate as compared with only sulfate addition was shown by qPCR analysis. Bacterial 16S rRNA gene analysis showed the presence of sulfate-reducing bacteria belonging to SEEP-SRB1. This is the first report that explicitly shows that AOM is associated with sulfate reduction in an enrichment culture of ANME-2a/b and AAA methanotrophs and SEEP-SRB1 sulfate reducers from a low-saline environment.We thank Douwe Bartstra (Vereniging tot Behoud van de Gasbronnen in Noord-Holland, The Netherlands), Carla Frijters (Paques BV, The Netherlands) and Teun Veuskens (Laboratory of Microbiology, WUR, The Netherlands) for sampling; Martin Meirink (Hoogheemraadschap Hollands Noorderkwartier, The Netherlands) for physicochemical data; Freek van Sambeek for providing Figure 1; Lennart Kleinjans (Laboratory of Microbiology, WUR, The Netherlands) for help with pyrosequencing analysis, Irene Sánchez-Andrea (Laboratory of Microbiology, WUR, The Netherlands) for proof-reading and Katharina Ettwig (Department of Microbiology, Radboud University Nijmegen, The Netherlands) for providing M. oxyfera DNA. We want to thank all anonymous reviewers for valuable contributions. This research is supported by the Dutch Technology Foundation STW (project 10711), which is part of the Netherlands Organization for Scientific Research (NWO), and which is partly funded by the Ministry of Economic Affairs. Research of AJMS is supported by ERC grant (project 323009) and the Gravitation grant (project 024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Science Foundation (NWO)

Role of X11 and ubiquilin as In Vivo Regulators of the Amyloid Precursor Protein in Drosophila

The Amyloid Precursor Protein (APP) undergoes sequential proteolytic cleavages through the action of β- and γ-secretase, which result in the generation of toxic β-amyloid (Aβ) peptides and a C-terminal fragment consisting of the intracellular domain of APP (AICD). Mutations leading to increased APP levels or alterations in APP cleavage cause familial Alzheimer's disease (AD). Thus, identification of factors that regulate APP steady state levels and/or APP cleavage by γ-secretase is likely to provide insight into AD pathogenesis. Here, using transgenic flies that act as reporters for endogenous γ-secretase activity and/or APP levels (GAMAREP), and for the APP intracellular domain (AICDREP), we identified mutations in X11L and ubiquilin (ubqn) as genetic modifiers of APP. Human homologs of both X11L (X11/Mint) and Ubqn (UBQLN1) have been implicated in AD pathogenesis. In contrast to previous reports, we show that overexpression of X11L or human X11 does not alter γ-secretase cleavage of APP or Notch, another γ-secretase substrate. Instead, expression of either X11L or human X11 regulates APP at the level of the AICD, and this activity requires the phosphotyrosine binding (PTB) domain of X11. In contrast, Ubqn regulates the levels of APP: loss of ubqn function leads to a decrease in the steady state levels of APP, while increased ubqn expression results in an increase in APP levels. Ubqn physically binds to APP, an interaction that depends on its ubiquitin-associated (UBA) domain, suggesting that direct physical interactions may underlie Ubqn-dependent regulation of APP. Together, our studies identify X11L and Ubqn as in vivo regulators of APP. Since increased expression of X11 attenuates Aβ production and/or secretion in APP transgenic mice, but does not act on γ-secretase directly, X11 may represent an attractive therapeutic target for AD

Long-range evolutionary constraints reveal cis-regulatory interactions on the human X chromosome

Enhancers can regulate the transcription of genes over long genomic distances. This is thought to lead to selection against genomic rearrangements within such regions that may disrupt this functional linkage. Here we test this concept experimentally using the human X chromosome. We describe a scoring method to identify evolutionary maintenance of linkage between conserved noncoding elements and neighbouring genes. Chromatin marks associated with enhancer function are strongly correlated with this linkage score. We test >1,000 putative enhancers by transgenesis assays in zebrafish to ascertain the identity of the target gene. The majority of active enhancers drive a transgenic expression in a pattern consistent with the known expression of a linked gene. These results show that evolutionary maintenance of linkage is a reliable predictor of an enhancer's function, and provide new information to discover the genetic basis of diseases caused by the mis-regulation of gene expression

Genetical genomics of growth in a chicken model

Background: The genetics underlying body mass and growth are key to understanding a wide range of topics in biology, both evolutionary and developmental. Body mass and growth traits are affected by many genetic variants of small effect. This complicates genetic mapping of growth and body mass. Experimental intercrosses between individuals from divergent populations allows us to map naturally occurring genetic variants for selected traits, such as body mass by linkage mapping. By simultaneously measuring traits and intermediary molecular phenotypes, such as gene expression, one can use integrative genomics to search for potential causative genes. Results: In this study, we use linkage mapping approach to map growth traits (N = 471) and liver gene expression (N = 130) in an advanced intercross of wild Red Junglefowl and domestic White Leghorn layer chickens. We find 16 loci for growth traits, and 1463 loci for liver gene expression, as measured by microarrays. Of these, the genes TRAK1, OSBPL8, YEATS4, CEP55, and PIP4K2B are identified as strong candidates for growth loci in the chicken. We also show a high degree of sex-specific gene-regulation, with almost every gene expression locus exhibiting sex-interactions. Finally, several trans-regulatory hotspots were found, one of which coincides with a major growth locus. Conclusions: These findings not only serve to identify several strong candidates affecting growth, but also show how sex-specificity and local gene-regulation affect growth regulation in the chicken.Funding Agencies|Carl Tryggers Stiftelse; Swedish Research Council (VR); Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS); Linkoping University Neuro-network; European Research Council [GENEWELL 322206]</p