CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping.

Broad-scale protein-protein interaction mapping is a major challenge given the cost, time, and sensitivity constraints of existing technologies. Here, we present a massively multiplexed yeast two-hybrid method, CrY2H-seq, which uses a Cre recombinase interaction reporter to intracellularly fuse the coding sequences of two interacting proteins and next-generation DNA sequencing to identify these interactions en masse. We applied CrY2H-seq to investigate sparsely annotated Arabidopsis thaliana transcription factors interactions. By performing ten independent screens testing a total of 36 million binary interaction combinations, and uncovering a network of 8,577 interactions among 1,453 transcription factors, we demonstrate CrY2H-seq's improved screening capacity, efficiency, and sensitivity over those of existing technologies. The deep-coverage network resource we call AtTFIN-1 recapitulates one-third of previously reported interactions derived from diverse methods, expands the number of known plant transcription factor interactions by three-fold, and reveals previously unknown family-specific interaction module associations with plant reproductive development, root architecture, and circadian coordination

The phosphatase laforin crosses evolutionary boundaries and links carbohydrate metabolism to neuronal disease

Lafora disease (LD) is a progressive myoclonic epilepsy resulting in severe neurodegeneration followed by death. A hallmark of LD is the accumulation of insoluble polyglucosans called Lafora bodies (LBs). LD is caused by mutations in the gene encoding the phosphatase laforin, which reportedly exists solely in vertebrates. We utilized a bioinformatics screen to identify laforin orthologues in five protists. These protists evolved from a progenitor red alga and synthesize an insoluble carbohydrate whose composition closely resembles LBs. Furthermore, we show that the kingdom Plantae, which lacks laforin, possesses a protein with laforin-like properties called starch excess 4 (SEX4). Mutations in the Arabidopsis thaliana SEX4 gene results in a starch excess phenotype reminiscent of LD. We demonstrate that Homo sapiens laforin complements the sex4 phenotype and propose that laforin and SEX4 are functional equivalents. Finally, we show that laforins and SEX4 dephosphorylate a complex carbohydrate and form the only family of phosphatases with this activity. These results provide a molecular explanation for the etiology of LD

Exploring the transcriptional landscape of plant circadian rhythms using genome tiling arrays

BACKGROUND Organisms are able to anticipate changes in the daily environment with an internal oscillator know as the circadian clock. Transcription is an important mechanism in maintaining these oscillations. Here we explore, using whole genome tiling arrays, the extent of rhythmic expression patterns genome-wide, with an unbiased analysis of coding and noncoding regions of the Arabidopsis genome. RESULTS As in previous studies, we detected a circadian rhythm for approximately 25% of the protein coding genes in the genome. With an unbiased interrogation of the genome, extensive rhythmic introns were detected predominantly in phase with adjacent rhythmic exons, creating a transcript that, if translated, would be expected to produce a truncated protein. In some cases, such as the MYB transcription factor AT2G20400, an intron was found to exhibit a circadian rhythm while the remainder of the transcript was otherwise arrhythmic. In addition to several known noncoding transcripts, including microRNA, trans-acting short interfering RNA, and small nucleolar RNA, greater than one thousand intergenic regions were detected as circadian clock regulated, many of which have no predicted function, either coding or noncoding. Nearly 7% of the protein coding genes produced rhythmic antisense transcripts, often for genes whose sense strand was not similarly rhythmic. CONCLUSIONS This study revealed widespread circadian clock regulation of the Arabidopsis genome extending well beyond the protein coding transcripts measured to date. This suggests a greater level of structural and temporal dynamics than previously known

Exploring \pp scattering in the \1N picture

In the large $N_c$ approximation to $QCD$, the leading \pp scattering amplitude is expressed as the sum of an infinite number of tree diagrams. We investigate the possibility that an adequate approximation at energies up to somewhat more than one $GeV$ can be made by keeping diagrams which involve the exchange of resonances in this energy range in addition to the simplest chiral contact terms. In this approach crossing symmetry is automatic but individual terms tend to drastically violate partial wave unitarity. We first note that the introduction of the $\rho$ meson in a chirally invariant manner substantially delays the onset of drastic unitarity violation which would be present for the {\it current algebra} term alone. This suggests a possibility of local (in energy) cancellation which we then explore in a phenomenological way. We include exchanges of leading resonances up to the $1.3 GeV$ region. However, unitarity requires more structure which we model by a four derivative contact term or by a low lying scalar resonance which is presumably subleading in the \1N expansion, but may nevertheless be important. The latter two flavor model gives a reasonable description of the phase shift $\delta^0_0$ up until around $860 MeV$, before the effects associated which the $K\bar{K}$ threshold come into play.Comment: 27 LaTex pages + 13 figures (also available in hard-copy

Rapid and High-Throughput pan-Orthopoxvirus Detection and Identification using PCR and Mass Spectrometry

The genus Orthopoxvirus contains several species of related viruses, including the causative agent of smallpox (Variola virus). In addition to smallpox, several other members of the genus are capable of causing human infection, including monkeypox, cowpox, and other zoonotic rodent-borne poxviruses. Therefore, a single assay that can accurately identify all orthopoxviruses could provide a valuable tool for rapid broad orthopovirus identification. We have developed a pan-Orthopoxvirus assay for identification of all members of the genus based on four PCR reactions targeting Orthopoxvirus DNA and RNA helicase and polymerase genes. The amplicons are detected using electrospray ionization-mass spectrometry (PCR/ESI-MS) on the Ibis T5000 system. We demonstrate that the assay can detect and identify a diverse collection of orthopoxviruses, provide sub-species information and characterize viruses from the blood of rabbitpox infected rabbits. The assay is sensitive at the stochastic limit of PCR and detected virus in blood containing approximately six plaque-forming units per milliliter from a rabbitpox virus-infected rabbit

Effects of Symmetry Breaking on the Strong and Electroweak Interactions of the Vector Nonet

Starting from a chiral invariant and quark line rule conserving Lagrangian of pseudoscalar and vector nonets we introduce first and second order symmetry breaking as well as quark line rule violating terms and fit the parameters, at tree level, to many strong and electroweak processes. A number of predictions are made. The electroweak interactions are included in a manifestly gauge invariant manner. The resulting symmetry breaking pattern is discussed in detail. Specifically, for the ``strong'' interactions, we study all the vector meson masses and V -> \phi \phi decays, including isotopic spin violations. In the electroweak sector we study the { rho^0 , omega , phi } -> e^+e^- decays, { pi^+ , K^+ , K^0 } ``charge radii'', K_{l3} ``slope factor'' and the overall e^+e^- -> pi^+ pi^- process. It is hoped that the resulting model may be useful as a reasonable description of low energy physics in the range up to about 1 GeV.Comment: 43 pages (LaTeX), 5 PostScript figures are included as uuencoded-compressed-tar file at the en

A Genome-Scale Resource for the Functional Characterization of Arabidopsis Transcription Factors

SummaryExtensive transcriptional networks play major roles in cellular and organismal functions. Transcript levels are in part determined by the combinatorial and overlapping functions of multiple transcription factors (TFs) bound to gene promoters. Thus, TF-promoter interactions provide the basic molecular wiring of transcriptional regulatory networks. In plants, discovery of the functional roles of TFs is limited by an increased complexity of network circuitry due to a significant expansion of TF families. Here, we present the construction of a comprehensive collection of Arabidopsis TFs clones created to provide a versatile resource for uncovering TF biological functions. We leveraged this collection by implementing a high-throughput DNA binding assay and identified direct regulators of a key clock gene (CCA1) that provide molecular links between different signaling modules and the circadian clock. The resources introduced in this work will significantly contribute to a better understanding of the transcriptional regulatory landscape of plant genomes

Global epigenomic reconfiguration during mammalian brain development

DNA methylation is implicated in mammalian brain development and plasticity underlying learning and memory. We report the genome-wide composition, patterning, cell specificity, and dynamics of DNA methylation at single-base resolution in human and mouse frontal cortex throughout their lifespan. Widespread methylome reconfiguration occurs during fetal to young adult development, coincident with synaptogenesis. During this period, highly conserved non-CG methylation (mCH) accumulates in neurons, but not glia, to become the dominant form of methylation in the human neuronal genome. Moreover, we found an mCH signature that identifies genes escaping X-chromosome inactivation. Last, whole-genome single-base resolution 5-hydroxymethylcytosine (hmC) maps revealed that hmC marks fetal brain cell genomes at putative regulatory regions that are CG-demethylated and activated in the adult brain and that CG demethylation at these hmC-poised loci depends on Tet2 activity

Molecular Criteria for Defining the Naive Human Pluripotent State.

Recent studies have aimed to convert cultured human pluripotent cells to a naive state, but it remains unclear to what extent the resulting cells recapitulate in vivo naive pluripotency. Here we propose a set of molecular criteria for evaluating the naive human pluripotent state by comparing it to the human embryo. We show that transcription of transposable elements provides a sensitive measure of the concordance between pluripotent stem cells and early human development. We also show that induction of the naive state is accompanied by genome-wide DNA hypomethylation, which is reversible except at imprinted genes, and that the X chromosome status resembles that of the human preimplantation embryo. However, we did not see efficient incorporation of naive human cells into mouse embryos. Overall, the different naive conditions we tested showed varied relationships to human embryonic states based on molecular criteria, providing a backdrop for future analysis of naive human pluripotency.This study was supported by grants from the Simons Foundation (SFLIFE #286977 to R.J) and in part by the NIH (RO1-CA084198) to R.J., from the Swiss National Science Foundation and the European Research Council (KRABnKAP, No. 268721) to D.T. The work in J.R.E’s laboratory was supported by the Howard Hughes Medical Institute and Gordon and Betty Moore Foundation (GBMF3034) and the Mary K. Chapman Foundation. J.R.E is an Investigator of the Howard Hughes Medical Institute. T.W.T. is supported by a Sir Henry Wellcome Postdoctoral Fellowship (098889/Z/12/Z), J.P. by a Foundation Bettencourt Award and by the Association pour la Recherche sur le Cancer (ARC), M.I. by a postdoctoral training grant from the Fonds de la Recherche en Santé du Québec. R.J. is co-founder of Fate Therapeutics and an adviser to Stemgent.This is the final version of the article. It first appeared from Cell Press via http://www.cell.com/cell-stem-cell/abstract/S1934-5909(16)30161-