Analysis of Thisbe and Pyramus functional domains reveals evidence for cleavage of Drosophila FGFs

Background: As important regulators of developmental and adult processes in metazoans, Fibroblast Growth Factor (FGF) proteins are potent signaling molecules whose activities must be tightly regulated. FGFs are known to play diverse roles in many processes, including mesoderm induction, branching morphogenesis, organ formation, wound healing and malignant transformation; yet much more remains to be learned about the mechanisms of regulation used to control FGF activity. Results: In this work, we conducted an analysis of the functional domains of two Drosophila proteins, Thisbe (Ths) and Pyramus (Pyr), which share homology with the FGF8 subfamily of ligands in vertebrates. Ths and Pyr proteins are secreted from Drosophila Schneider cells (S2) as smaller N-terminal fragments presumably as a result of intracellular proteolytic cleavage. Cleaved forms of Ths and Pyr can be detected in embryonic extracts as well. The FGF-domain is contained within the secreted ligand portion, and this domain alone is capable of functioning in the embryo when ectopically expressed. Through targeted ectopic expression experiments in which we assay the ability of full-length, truncated, and chimeric proteins to support cell differentiation, we find evidence that (1) the C-terminal domain of Pyr is retained inside the cell and does not seem to be required for receptor activation and (2) the C-terminal domain of Ths is secreted and, while also not required for receptor activation, this domain does plays a role in limiting the activity of Ths when present. Conclusions: We propose that differential protein processing may account for the previously observed inequalities in signaling capabilities between Ths and Pyr. While the regulatory mechanisms are likely complex, studies such as ours conducted in a tractable model system may be able to provide insights into how ligand processing regulates growth factor activity

Extending the family table: Insights from beyond vertebrates into the regulation of embryonic development by FGFs

Since the discovery of fibroblast growth factors (FGFs) much focus has been placed on elucidating the roles for each vertebrate FGF ligand, receptor, and regulating molecules in the context of vertebrate development, human disorders and cancer. Studies in human, mouse, frog, chick, and zebrafish have made great contributions to our understanding of the role of FGFs in specific processes. However, in recent years, as more genomes are sequenced, information is becoming available from many non-vertebrate models and a more complete picture of the FGF superfamily as a whole is emerging. In some cases, less redundancy in these FGF signaling systems may allow for more mechanistic insights. Studies in sea anemones have highlighted how ancient FGF signaling is and helped provide insight into the evolution of the FGF gene family. Work in nematodes has shown that different splice forms can be used for functional specificity in invertebrate FGF signaling. Comparing FGFs between urochordates and vertebrates as well as between different insect species reveals important clues into the process of gene loss, duplication and subfunctionalization of FGFs throughout evolution. Finally, comparing all members of the FGF ligand superfamily reveals variability in many properties, which may point to a feature of FGFs as being highly adaptable with regards to protein structure and signaling mechanism. Further studies on FGF signaling outside of vertebrates is likely to continue to complement work in vertebrates by contributing additional insights to the FGF field and providing unexpected information that could be used for medical applications

A quantitative reference transcriptome for Nematostella vectensis early embryonic development : a pipeline for de novo assembly in emerging model systems

© The Author(s), 2013. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in EvoDevo 4 (2013): 16, doi:10.1186/2041-9139-4-16.The de novo assembly of transcriptomes from short shotgun sequences raises challenges due to random and non-random sequencing biases and inherent transcript complexity. We sought to define a pipeline for de novo transcriptome assembly to aid researchers working with emerging model systems where well annotated genome assemblies are not available as a reference. To detail this experimental and computational method, we used early embryos of the sea anemone, Nematostella vectensis, an emerging model system for studies of animal body plan evolution. We performed RNA-seq on embryos up to 24 h of development using Illumina HiSeq technology and evaluated independent de novo assembly methods. The resulting reads were assembled using either the Trinity assembler on all quality controlled reads or both the Velvet and Oases assemblers on reads passing a stringent digital normalization filter. A control set of mRNA standards from the National Institute of Standards and Technology (NIST) was included in our experimental pipeline to invest our transcriptome with quantitative information on absolute transcript levels and to provide additional quality control. We generated >200 million paired-end reads from directional cDNA libraries representing well over 20 Gb of sequence. The Trinity assembler pipeline, including preliminary quality control steps, resulted in more than 86% of reads aligning with the reference transcriptome thus generated. Nevertheless, digital normalization combined with assembly by Velvet and Oases required far less computing power and decreased processing time while still mapping 82% of reads. We have made the raw sequencing reads and assembled transcriptome publically available. Nematostella vectensis was chosen for its strategic position in the tree of life for studies into the origins of the animal body plan, however, the challenge of reference-free transcriptome assembly is relevant to all systems for which well annotated gene models and independently verified genome assembly may not be available. To navigate this new territory, we have constructed a pipeline for library preparation and computational analysis for de novo transcriptome assembly. The gene models defined by this reference transcriptome define the set of genes transcribed in early Nematostella development and will provide a valuable dataset for further gene regulatory network investigations

Genome-wide identification of enhancer elements

We present a prospective genome-wide regulatory element database for the sea urchin embryo and the modified chromosome capture-related methodology used to create it. The method we developed is termed GRIP-seq for genome-wide regulatory element immunoprecipitation and combines features of chromosome conformation capture, chromatin immunoprecipitation, and paired-end next-generation sequencing with molecular steps that enrich for active cis-regulatory elements associated with basal transcriptional machinery. The first GRIP-seq database, available to the community, comes from S. purpuratus 24 hpf embryos and takes advantage of the extremely well-characterized cis-regulatory elements in this system for validation. In addition, using the GRIP-seq database, we identify and experimentally validate a novel, intronic cis-regulatory element at the onecut locus. We find GRIP-seq signal sensitively identifies active cis-regulatory elements with a high signal-to-noise ratio for both distal and intronic elements. This promising GRIP-seq protocol has the potential to address a rate-limiting step in resolving comprehensive, predictive network models in all systems

Hit and run versus long-term activation of PARP-1 by its different domains fine-tunes nuclear processes.

Poly(ADP-ribose) polymerase 1 (PARP-1) is a multidomain multifunctional nuclear enzyme involved in the regulation of the chromatin structure and transcription. PARP-1 consists of three functional domains: the N-terminal DNA-binding domain (DBD) containing three zinc fingers, the automodification domain (A), and the C-terminal domain, which includes the protein interacting WGR domain (W) and the catalytic (Cat) subdomain responsible for the poly(ADP ribosyl)ating reaction. The mechanisms coordinating the functions of these domains and determining the positioning of PARP-1 in chromatin remain unknown. Using multiple deletional isoforms of PARP-1, lacking one or another of its three domains, as well as consisting of only one of those domains, we demonstrate that different functions of PARP-1 are coordinated by interactions among these domains and their targets. Interaction between the DBD and damaged DNA leads to a short-term binding and activation of PARP-1. This hit and run activation of PARP-1 initiates the DNA repair pathway at a specific point. The long-term chromatin loosening required to sustain transcription takes place when the C-terminal domain of PARP-1 binds to chromatin by interacting with histone H4 in the nucleosome. This long-term activation of PARP-1 results in a continuous accumulation of pADPr, which maintains chromatin in the loosened state around a certain locus so that the transcription machinery has continuous access to DNA. Cooperation between the DBD and C-terminal domain occurs in response to heat shock (HS), allowing PARP-1 to scan chromatin for specific binding sites

Employing BAC-reporter constructs in the sea anemone Nematostella vectensis

Author Posting. © The Author(s), 2013. This is the author's version of the work. It is posted here by permission of Oxford University Press for personal use, not for redistribution. The definitive version was published in Integrative and Comparative Biology 53 (2013): 832-846, doi:10.1093/icb/ict091.Changes in the expression and function of genes drive evolutionary change. Comparing how genes are regulated in different species is therefore becoming an important part of evo-devo studies. A key tool for investigating the regulation of genes is represented by Bacterial Artificial Chromosomes-reporter constructs (BAC). BACs are large insert libraries, often >>100 kb, which thus capture the genomic sequences surrounding a gene of interest, including all, or nearly all, of the elements underpinning regulation. Recombinant BACs, containing a reporter gene in place of the endogenous coding sequence of genes, can be utilized to drive the expression of reporter genes under the regulatory control of the gene of interest while still embedded within its genomic context. Systematic deletions within the BAC reporter construct can be used to identify the minimal reporter in an unbiased way, avoiding the risk of overlooking regulatory elements that may be many kilobases away from the transcription start-site. Nematostella vectensis (Edwardsiidae, Anthozoa, Cnidaria) has become an important model in regenerative biology, ecology, and especially in studies of evo-devo and gene-regulatory networks due to its interesting phylogenetic position and amenability to molecular techniques. The increasing interest in this rising model system also led to a demand for methods that can be used to study the regulation of genes in Nematostella. Here we present our progress in employing BAC reporter constructs to visualize gene-expression in Nematostella. Using a new Nematostella-specific recombination cassette, we made nine different BAC reporter constructs. Although five BAC recombinants gave variable effects, three constructs, namely Nv-bra:eGFP::L10 BAC, Nv-dpp:eGFP::L10 BAC, and Nv-grm:eGFP::L10 BAC, delivered promising results. We show that these three constructs express the reporter gene eGFP in 10.4% – 17.2% of all analyzed larvae, out of which 26.2 – 41.9% express GFP in a mosaic fashion within the expected domain. In addition to the expression within the known domains, we also observed cases of misexpression of eGFP and examples that could represent actual expression outside the described domain. Furthermore, we deep-sequenced and assembled five different BACs containing Nv-chordin, Nv-foxa, Nv-dpp, Nv-wnta, and Nvwnt1, to improve assembly around these genes. The use of BAC reporter constructs will foster cis-regulatory analyses in Nematostella and thus help to improve our understanding of the regulatory network in this cnidarian system. Ultimately, this will advance the comparison of gene-regulation across species and lead to a much better understanding of evolutionary changes and novelties.2014-08-1

A framework for human microbiome research

A variety of microbial communities and their genes (the microbiome) exist throughout the human body, with fundamental roles in human health and disease. The National Institutes of Health (NIH)-funded Human Microbiome Project Consortium has established a population-scale framework to develop metagenomic protocols, resulting in a broad range of quality-controlled resources and data including standardized methods for creating, processing and interpreting distinct types of high-throughput metagenomic data available to the scientific community. Here we present resources from a population of 242 healthy adults sampled at 15 or 18 body sites up to three times, which have generated 5,177 microbial taxonomic profiles from 16S ribosomal RNA genes and over 3.5 terabases of metagenomic sequence so far. In parallel, approximately 800 reference strains isolated from the human body have been sequenced. Collectively, these data represent the largest resource describing the abundance and variety of the human microbiome, while providing a framework for current and future studies

Structure, function and diversity of the healthy human microbiome

Author Posting. © The Authors, 2012. This article is posted here by permission of Nature Publishing Group. The definitive version was published in Nature 486 (2012): 207-214, doi:10.1038/nature11234.Studies of the human microbiome have revealed that even healthy individuals differ remarkably in the microbes that occupy habitats such as the gut, skin and vagina. Much of this diversity remains unexplained, although diet, environment, host genetics and early microbial exposure have all been implicated. Accordingly, to characterize the ecology of human-associated microbial communities, the Human Microbiome Project has analysed the largest cohort and set of distinct, clinically relevant body habitats so far. We found the diversity and abundance of each habitat’s signature microbes to vary widely even among healthy subjects, with strong niche specialization both within and among individuals. The project encountered an estimated 81–99% of the genera, enzyme families and community configurations occupied by the healthy Western microbiome. Metagenomic carriage of metabolic pathways was stable among individuals despite variation in community structure, and ethnic/racial background proved to be one of the strongest associations of both pathways and microbes with clinical metadata. These results thus delineate the range of structural and functional configurations normal in the microbial communities of a healthy population, enabling future characterization of the epidemiology, ecology and translational applications of the human microbiome.This research was supported in part by National Institutes of Health grants U54HG004969 to B.W.B.; U54HG003273 to R.A.G.; U54HG004973 to R.A.G., S.K.H. and J.F.P.; U54HG003067 to E.S.Lander; U54AI084844 to K.E.N.; N01AI30071 to R.L.Strausberg; U54HG004968 to G.M.W.; U01HG004866 to O.R.W.; U54HG003079 to R.K.W.; R01HG005969 to C.H.; R01HG004872 to R.K.; R01HG004885 to M.P.; R01HG005975 to P.D.S.; R01HG004908 to Y.Y.; R01HG004900 to M.K.Cho and P. Sankar; R01HG005171 to D.E.H.; R01HG004853 to A.L.M.; R01HG004856 to R.R.; R01HG004877 to R.R.S. and R.F.; R01HG005172 to P. Spicer.; R01HG004857 to M.P.; R01HG004906 to T.M.S.; R21HG005811 to E.A.V.; M.J.B. was supported by UH2AR057506; G.A.B. was supported by UH2AI083263 and UH3AI083263 (G.A.B., C. N. Cornelissen, L. K. Eaves and J. F. Strauss); S.M.H. was supported by UH3DK083993 (V. B. Young, E. B. Chang, F. Meyer, T. M. S., M. L. Sogin, J. M. Tiedje); K.P.R. was supported by UH2DK083990 (J. V.); J.A.S. and H.H.K. were supported by UH2AR057504 and UH3AR057504 (J.A.S.); DP2OD001500 to K.M.A.; N01HG62088 to the Coriell Institute for Medical Research; U01DE016937 to F.E.D.; S.K.H. was supported by RC1DE0202098 and R01DE021574 (S.K.H. and H. Li); J.I. was supported by R21CA139193 (J.I. and D. S. Michaud); K.P.L. was supported by P30DE020751 (D. J. Smith); Army Research Office grant W911NF-11-1-0473 to C.H.; National Science Foundation grants NSF DBI-1053486 to C.H. and NSF IIS-0812111 to M.P.; The Office of Science of the US Department of Energy under Contract No. DE-AC02-05CH11231 for P.S. C.; LANL Laboratory-Directed Research and Development grant 20100034DR and the US Defense Threat Reduction Agency grants B104153I and B084531I to P.S.C.; Research Foundation - Flanders (FWO) grant to K.F. and J.Raes; R.K. is an HHMI Early Career Scientist; Gordon&BettyMoore Foundation funding and institutional funding fromthe J. David Gladstone Institutes to K.S.P.; A.M.S. was supported by fellowships provided by the Rackham Graduate School and the NIH Molecular Mechanisms in Microbial Pathogenesis Training Grant T32AI007528; a Crohn’s and Colitis Foundation of Canada Grant in Aid of Research to E.A.V.; 2010 IBM Faculty Award to K.C.W.; analysis of the HMPdata was performed using National Energy Research Scientific Computing resources, the BluBioU Computational Resource at Rice University

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements