MYC acetylated lysine residues drive oncogenic cell transformation and regulate select genetic programs for cell adhesion-independent growth and survival.

The MYC oncogenic transcription factor is acetylated by the p300 and GCN5 histone acetyltransferases. The significance of MYC acetylation and the functions of specific acetylated lysine (AcK) residues have remained unclear. Here, we show that the major p300-acetylated K148(149) and K157(158) sites in human (or mouse) MYC and the main GCN5-acetylated K323 residue are reversibly acetylated in various malignant and nonmalignant cells. Oncogenic overexpression of MYC enhances its acetylation and alters the regulation of site-specific acetylation by proteasome and deacetylase inhibitors. Acetylation of MYC at different K residues differentially affects its stability in a cell type-dependent manner. Lysine-to-arginine substitutions indicate that although none of the AcK residues is required for MYC stimulation of adherent cell proliferation, individual AcK sites have gene-specific functions controlling select MYC-regulated processes in cell adhesion, contact inhibition, apoptosis, and/or metabolism and are required for the malignant cell transformation activity of MYC. Each AcK site is required for anchorage-independent growth of MYC-overexpressing cells in vitro, and both the AcK148(149) and AcK157(158) residues are also important for the tumorigenic activity of MYC transformed cells in vivo. The MYC AcK site-specific signaling pathways identified may offer new avenues for selective therapeutic targeting of MYC oncogenic activities.

signatureSearch: environment for gene expression signature searching and functional interpretation.

signatureSearch is an R/Bioconductor package that integrates a suite of existing and novel algorithms into an analysis environment for gene expression signature (GES) searching combined with functional enrichment analysis (FEA) and visualization methods to facilitate the interpretation of the search results. In a typical GES search (GESS), a query GES is searched against a database of GESs obtained from large numbers of measurements, such as different genetic backgrounds, disease states and drug perturbations. Database matches sharing correlated signatures with the query indicate related cellular responses frequently governed by connected mechanisms, such as drugs mimicking the expression responses of a disease. To identify which processes are predominantly modulated in the GESS results, we developed specialized FEA methods combined with drug-target network visualization tools. The provided analysis tools are useful for studying the effects of genetic, chemical and environmental perturbations on biological systems, as well as searching single cell GES databases to identify novel network connections or cell types. The signatureSearch software is unique in that it provides access to an integrated environment for GESS/FEA routines that includes several novel search and enrichment methods, efficient data structures, and access to pre-built GES databases, and allowing users to work with custom databases.

Transcriptome-wide microRNA and target dynamics in the fat body during the gonadotrophic cycle of Aedes aegypti.

The mosquito Aedes aegypti is a major vector of numerous viral diseases, because it requires a blood meal to facilitate egg development. The fat body, a counterpart of mammalian liver and adipose tissues, is the metabolic center, playing a key role in reproduction. Therefore, understanding of regulatory networks controlling its functions is critical, and the role of microRNAs (miRNAs) in the process is largely unknown. We aimed to explore miRNA expression and potential targets in the female fat body of Ae. aegypti, as well as their changes postblood meal (PBM). Small RNA library analysis revealed five unique miRNA patterns sequentially expressed at five sampled time points, likely responding to, and affecting, waves of upstream hormonal signals and gene expression in the same period. To link miRNA identities with downstream targets, transcriptome-wide mRNA 3' UTR interaction sites were experimentally determined at 72 h posteclosion and 24 h PBM through Argonaute 1 cross-linking and immunoprecipitation followed by high-throughput sequencing. Several target sites were validated by means of in vitro luciferase assays with wild-type and mutated 3' UTRs for six miRNA families. With established transgenic lines, consistent results were observed with spatiotemporal knockdown of miR-8 and luciferase assays. We further investigated miRNAs potentially regulating various physiological processes based on Clusters of Orthologous Groups functional categories. Hence, the present work comprehensively elucidated miRNA expression and target dynamics in the female mosquito fat body, providing a solid foundation for future functional studies of miRNA regulation during the gonadotrophic cycle.

Hairy and Groucho mediate the action of juvenile hormone receptor Methoprene-tolerant in gene repression.

The arthropod-specific juvenile hormone (JH) controls numerous essential functions. Its involvement in gene activation is known to be mediated by the transcription factor Methoprene-tolerant (Met), which turns on JH-controlled genes by directly binding to E-box-like motifs in their regulatory regions. However, it remains unclear how JH represses genes. We used the Aedes aegypti female mosquito, in which JH is necessary for reproductive maturation, to show that a repressor, Hairy, is required for the gene-repressive action of JH and Met. The RNA interference (RNAi) screen for Met and Hairy in the Aedes female fat body revealed a large cohort of Met- and Hairy-corepressed genes. Analysis of selected genes from this cohort demonstrated that they are repressed by JH, but RNAi of either Met or Hairy renders JH ineffective in repressing these genes in an in vitro fat-body culture assay. Moreover, this JH action was prevented by the addition of the translational inhibitor cycloheximide (CHX) to the culture, indicating the existence of an indirect regulatory hierarchy. The lack of Hairy protein in the CHX-treated tissue was verified using immunoblot analysis, and the upstream regions of Met/Hairy-corepressed genes were shown to contain common binding motifs that interact with Hairy. Groucho (gro) RNAi silencing phenocopied the effect of Hairy RNAi knockdown, indicating that it is involved in the JH/Met/Hairy hierarchy. Finally, the requirement of Hairy and Gro for gene repression was confirmed in a cell transfection assay. Thus, our study has established that Hairy and its cofactor Gro mediate the repressive function of JH and Met.

WUSCHEL protein movement mediates stem cell homeostasis in the Arabidopsis shoot apex.

WUSCHEL (WUS) is a homeodomain transcription factor produced in cells of the niche/organizing center (OC) of shoot apical meristems. WUS specifies stem cell fate and also restricts its own levels by activating a negative regulator, CLAVATA3 (CLV3), in adjacent cells of the central zone (CZ). Here we show that the WUS protein, after being synthesized in cells of the OC, migrates into the CZ, where it activates CLV3 transcription by binding to its promoter elements. Using a computational model, we show that maintenance of the WUS gradient is essential to regulate stem cell number. Migration of a stem cell-inducing transcription factor into adjacent cells to activate a negative regulator, thereby restricting its own accumulation, is a theme that is unique to plant stem cell niches.

Orchestrating high-throughput genomic analysis with Bioconductor.

Bioconductor is an open-source, open-development software project for the analysis and comprehension of high-throughput data in genomics and molecular biology. The project aims to enable interdisciplinary research, collaboration and rapid development of scientific software. Based on the statistical programming language R, Bioconductor comprises 934 interoperable packages contributed by a large, diverse community of scientists. Packages cover a range of bioinformatic and statistical applications. They undergo formal initial review and continuous automated testing. We present an overview for prospective users and contributors.

Alcohol Consumption Modulates Host Defense in Rhesus Macaques by Altering Gene Expression in Circulating Leukocytes.

Several lines of evidence indicate that chronic alcohol use disorder leads to increased susceptibility to several viral and bacterial infections, whereas moderate alcohol consumption decreases the incidence of colds and improves immune responses to some pathogens. In line with these observations, we recently showed that heavy ethanol intake (average blood ethanol concentrations > 80 mg/dl) suppressed, whereas moderate alcohol consumption (blood ethanol concentrations < 50 mg/dl) enhanced, T and B cell responses to modified vaccinia Ankara vaccination in a nonhuman primate model of voluntary ethanol consumption. To uncover the molecular basis for impaired immunity with heavy alcohol consumption and enhanced immune response with moderate alcohol consumption, we performed a transcriptome analysis using PBMCs isolated on day 7 post-modified vaccinia Ankara vaccination, the earliest time point at which we detected differences in T cell and Ab responses. Overall, chronic heavy alcohol consumption reduced the expression of immune genes involved in response to infection and wound healing and increased the expression of genes associated with the development of lung inflammatory disease and cancer. In contrast, chronic moderate alcohol consumption upregulated the expression of genes involved in immune response and reduced the expression of genes involved in cancer. To uncover mechanisms underlying the alterations in PBMC transcriptomes, we profiled the expression of microRNAs within the same samples. Chronic heavy ethanol consumption altered the levels of several microRNAs involved in cancer and immunity and known to regulate the expression of mRNAs differentially expressed in our data set.

Regulation of Gene Expression Patterns in Mosquito Reproduction.

In multicellular organisms, development, growth and reproduction require coordinated expression of numerous functional and regulatory genes. Insects, in addition to being the most speciose animal group with enormous biological and economical significance, represent outstanding model organisms for studying regulation of synchronized gene expression due to their rapid development and reproduction. Disease-transmitting female mosquitoes have adapted uniquely for ingestion and utilization of the huge blood meal required for swift reproductive events to complete egg development within a 72-h period. We investigated the network of regulatory factors mediating sequential gene expression in the fat body, a multifunctional organ analogous to the vertebrate liver and adipose tissue, of the female Aedes aegypti mosquito. Transcriptomic and bioinformatics analyses revealed that ~7500 transcripts are differentially expressed in four sequential waves during the 72-h reproductive period. A combination of RNA-interference gene-silencing and in-vitro organ culture identified the major regulators for each of these waves. Amino acids (AAs) regulate the first wave of gene activation between 3 h and 12 h post-blood meal (PBM). During the second wave, between 12 h and 36 h, most genes are highly upregulated by a synergistic action of AAs, 20-hydroxyecdysone (20E) and the Ecdysone-Receptor (EcR). Between 36 h and 48 h, the third wave of gene activation-regulated mainly by HR3-occurs. Juvenile Hormone (JH) and its receptor Methoprene-Tolerant (Met) are major regulators for the final wave between 48 h and 72 h. Each of these key regulators also has repressive effects on one or more gene sets. Our study provides a better understanding of the complexity of the regulatory mechanisms related to temporal coordination of gene expression during reproduction. We have detected the novel function of 20E/EcR responsible for transcriptional repression. This study also reveals the previously unidentified large-scale effects of HR3 and JH/Met on transcriptional regulation during the termination of vitellogenesis and remodeling of the fat body.

A high-resolution gene expression map of the Arabidopsis shoot meristem stem cell niche.

The shoot apical meristem (SAM) acts as a reservoir for stem cells. The central zone (CZ) harbors stem cells. The stem cell progenitors differentiate in the adjacent peripheral zone and in the rib meristem located just beneath the CZ. The SAM is further divided into distinct clonal layers: the L1 epidermal, L2 sub-epidermal and L3 layers. Collectively, SAMs are complex structures that consist of cells of different clonal origins that are organized into functional domains. By employing fluorescence-activated cell sorting, we have generated gene expression profiles of ten cell populations that belong to different clonal layers as well as domains along the central and peripheral axis. Our work reveals that cells in distinct clonal layers exhibit greater diversity in gene expression and greater transcriptional complexity than clonally related cell types in the central and peripheral axis. Assessment of molecular functions and biological processes reveals that epidermal cells express genes involved in pathogen defense: the L2 layer cells express genes involved in DNA repair pathways and telomere maintenance, and the L3 layers express transcripts involved in ion balance and salt tolerance besides photosynthesis. Strikingly, the stem cell-enriched transcriptome comprises very few hormone-responsive transcripts. In addition to providing insights into the expression profiles of hundreds of transcripts, the data presented here will act as a resource for reverse genetic analysis and will be useful in deciphering molecular pathways involved in cell type specification and their functions.

Acute Simian Varicella Virus Infection Causes Robust and Sustained Changes in Gene Expression in the Sensory Ganglia.

Primary infection with varicella-zoster virus (VZV), a neurotropic alphaherpesvirus, results in varicella. VZV establishes latency in the sensory ganglia and can reactivate later in life to cause herpes zoster. The relationship between VZV and its host during acute infection in the sensory ganglia is not well understood due to limited access to clinical specimens. Intrabronchial inoculation of rhesus macaques with simian varicella virus (SVV) recapitulates the hallmarks of VZV infection in humans. We leveraged this animal model to characterize the host-pathogen interactions in the ganglia during both acute and latent infection by measuring both viral and host transcriptomes on days postinfection (dpi) 3, 7, 10, 14, and 100. SVV DNA and transcripts were detected in sensory ganglia 3 dpi, before the appearance of rash. CD4 and CD8 T cells were also detected in the sensory ganglia 3 dpi. Moreover, lung-resident T cells isolated from the same animals 3 dpi also harbored SVV DNA and transcripts, suggesting that T cells may be responsible for trafficking SVV to the ganglia. Transcriptome sequencing (RNA-Seq) analysis showed that cessation of viral transcription 7 dpi coincides with a robust antiviral innate immune response in the ganglia. Interestingly, a significant number of genes that play a critical role in nervous system development and function remained downregulated into latency. These studies provide novel insights into host-pathogen interactions in the sensory ganglia during acute varicella and demonstrate that SVV infection results in profound and sustained changes in neuronal gene expression. IMPORTANCE: Many aspects of VZV infection of sensory ganglia remain poorly understood, due to limited access to human specimens and the fact that VZV is strictly a human virus. Infection of rhesus macaques with simian varicella virus (SVV), a homolog of VZV, provides a robust model of the human disease. Using this model, we show that SVV reaches the ganglia early after infection, most likely by T cells, and that the induction of a robust innate immune response correlates with cessation of virus transcription. We also report significant changes in the expression of genes that play an important role in neuronal function. Importantly, these changes persist long after viral replication ceases. Given the homology between SVV and VZV, and the genetic and physiological similarities between rhesus macaques and humans, our results provide novel insight into the interactions between VZV and its human host and explain some of the neurological consequences of VZV infection.

Transcriptomes of Eight Arabidopsis thaliana Accessions Reveal Core Conserved, Genotype- and Organ-Specific Responses to Flooding Stress.

Climate change has increased the frequency and severity of flooding events, with significant negative impact on agricultural productivity. These events often submerge plant aerial organs and roots, limiting growth and survival due to a severe reduction in light reactions and gas exchange necessary for photosynthesis and respiration, respectively. To distinguish molecular responses to the compound stress imposed by submergence, we investigated transcriptomic adjustments to darkness in air and under submerged conditions using eight Arabidopsis (Arabidopsis thaliana) accessions differing significantly in sensitivity to submergence. Evaluation of root and rosette transcriptomes revealed an early transcriptional and posttranscriptional response signature that was conserved primarily across genotypes, although flooding susceptibility-associated and genotype-specific responses also were uncovered. Posttranscriptional regulation encompassed darkness- and submergence-induced alternative splicing of transcripts from pathways involved in the alternative mobilization of energy reserves. The organ-specific transcriptome adjustments reflected the distinct physiological status of roots and shoots. Root-specific transcriptome changes included marked up-regulation of chloroplast-encoded photosynthesis and redox-related genes, whereas those of the rosette were related to the regulation of development and growth processes. We identified a novel set of tolerance genes, recognized mainly by quantitative differences. These included a transcriptome signature of more pronounced gluconeogenesis in tolerant accessions, a response that included stress-induced alternative splicing. This study provides organ-specific molecular resolution of genetic variation in submergence responses involving interactions between darkness and low-oxygen constraints of flooding stress and demonstrates that early transcriptome plasticity, including alternative splicing, is associated with the ability to cope with a compound environmental stress.

Translational dynamics revealed by genome-wide profiling of ribosome footprints in Arabidopsis.

Translational regulation contributes to plasticity in metabolism and growth that enables plants to survive in a dynamic environment. Here, we used the precise mapping of ribosome footprints (RFs) on mRNAs to investigate translational regulation under control and sublethal hypoxia stress conditions in seedlings of Arabidopsis thaliana. Ribosomes were obtained by differential centrifugation or immunopurification and were digested with RNase I to generate footprint fragments that were deep-sequenced. Comparison of RF number and position on genic regions with fragmented total and polysomal mRNA illuminated numerous aspects of posttranscriptional and translational control under both growth conditions. When seedlings were oxygen-deprived, the frequency of ribosomes at the start codon was reduced, consistent with a global decline in initiation of translation. Hypoxia-up-regulated gene transcripts increased in polysome complexes during the stress, but the number of ribosomes per transcript relative to normoxic conditions was not enhanced. On the other hand, many mRNAs with limited change in steady-state abundance had significantly fewer ribosomes but with an overall similar distribution under hypoxia, consistent with restriction of initiation rather than elongation of translation. RF profiling also exposed the inhibitory effect of upstream ORFs on the translation of downstream protein-coding regions under normoxia, which was further modulated by hypoxia. The data document translation of alternatively spliced mRNAs and expose ribosome association with some noncoding RNAs. Altogether, we present an experimental approach that illuminates prevalent and nuanced regulation of protein synthesis under optimal and energy-limiting conditions.

systemPipeR: NGS workflow and report generation environment.

BACKGROUND: Next-generation sequencing (NGS) has revolutionized how research is carried out in many areas of biology and medicine. However, the analysis of NGS data remains a major obstacle to the efficient utilization of the technology, as it requires complex multi-step processing of big data demanding considerable computational expertise from users. While substantial effort has been invested on the development of software dedicated to the individual analysis steps of NGS experiments, insufficient resources are currently available for integrating the individual software components within the widely used R/Bioconductor environment into automated workflows capable of running the analysis of most types of NGS applications from start-to-finish in a time-efficient and reproducible manner. RESULTS: To address this need, we have developed the R/Bioconductor package systemPipeR. It is an extensible environment for both building and running end-to-end analysis workflows with automated report generation for a wide range of NGS applications. Its unique features include a uniform workflow interface across different NGS applications, automated report generation, and support for running both R and command-line software on local computers and computer clusters. A flexible sample annotation infrastructure efficiently handles complex sample sets and experimental designs. To simplify the analysis of widely used NGS applications, the package provides pre-configured workflows and reporting templates for RNA-Seq, ChIP-Seq, VAR-Seq and Ribo-Seq. Additional workflow templates will be provided in the future. CONCLUSIONS: systemPipeR accelerates the extraction of reproducible analysis results from NGS experiments. By combining the capabilities of many R/Bioconductor and command-line tools, it makes efficient use of existing software resources without limiting the user to a set of predefined methods or environments. systemPipeR is freely available for all common operating systems from Bioconductor ( http://bioconductor.org/packages/devel/systemPipeR ).

A novel virus from Macrosiphum euphorbiae with similarities to members of the family Flaviviridae.

A virus with a large genome was identified in the transcriptome of the potato aphid (Macrosiphum euphorbiae) and was named Macrosiphum euphorbiae virus 1 (MeV-1). The MeV-1 genome is 22 780 nt in size, including 3' and 5' non-coding regions, with a single large ORF encoding a putative polyprotein of 7333 aa. The C-terminal region of the predicted MeV-1 polyprotein contained sequences with similarities to helicase, methyltransferase and RNA-dependent RNA polymerase (RdRp) motifs, while the N-terminal region lacked any motifs including structural proteins. Phylogenetic analysis of the helicase placed MeV-1 close to pestiviruses, while the RdRp region placed it close to pestiviruses and flaviviruses, suggesting MeV-1 has a positive-polarity ssRNA genome and is a member of the family Flaviviridae. Since the MeV-1 genome is predicted to contain a methyltransferase, a gene present typically in flaviviruses but not pestiviruses, MeV-1 is likely a member of the genus Flavivirus. MeV-1 was present in nymphal and adult stages of the aphid, aphid saliva and plant tissues fed upon by aphids. However, the virus was unable to multiply and spread in tomato plants. In addition, dsRNA, the replication intermediate of RNA viruses, was isolated from virus-infected M. euphorbiae and not from tomato plants infested with the aphid. Furthermore, nymphs laid without exposure to infected plants harboured the virus, indicating that MeV-1 is an aphid-infecting virus likely transmitted transovarially. The virus was present in M. euphorbiae populations from Europe but not from North America and was absent in all other aphid species tested.

bioassayR: Cross-Target Analysis of Small Molecule Bioactivity.

Despite a large and rapidly growing body of small molecule bioactivity screens available in the public domain, systematic leverage of the data to assess target druggability and compound selectivity has been confounded by a lack of suitable cross-target analysis software. We have developed bioassayR, a computational tool that enables simultaneous analysis of thousands of bioassay experiments performed over a diverse set of compounds and biological targets. Unique features include support for large-scale cross-target analyses of both public and custom bioassays, generation of high throughput screening fingerprints (HTSFPs), and an optional preloaded database that provides access to a substantial portion of publicly available bioactivity data. bioassayR is implemented as an open-source R/Bioconductor package available from https://bioconductor.org/packages/bioassayR/ .

Juvenile hormone and its receptor, methoprene-tolerant, control the dynamics of mosquito gene expression.

Juvenile hormone III (JH) plays a key role in regulating the reproduction of female mosquitoes. Microarray time-course analysis revealed dynamic changes in gene expression during posteclosion (PE) development in the fat body of female Aedes aegypti. Hierarchical clustering identified three major gene clusters: 1,843 early-PE (EPE) genes maximally expressed at 6 h PE, 457 mid-PE (MPE) genes at 24 h PE, and 1,815 late-PE (LPE) genes at 66 h PE. The RNAi microarray screen for the JH receptor Methoprene-tolerant (Met) showed that 27% of EPE and 40% of MPE genes were up-regulated whereas 36% of LPE genes were down-regulated in the absence of this receptor. Met repression of EPE and MPE and activation of LPE genes were validated by an in vitro fat-body culture experiment using Met RNAi. Sequence motif analysis revealed the consensus for a 9-mer Met-binding motif, CACG(C)/TG(A)/G(T)/AG. Met-binding motif variants were overrepresented within the first 300 bases of the promoters of Met RNAi-down-regulated (LPE) genes but not in Met RNAi-up-regulated (EPE) genes. EMSAs using a combination of mutational and anti-Met antibody supershift analyses confirmed the binding properties of the Met consensus motif variants. There was a striking temporal separation of expression profiles among major functional gene groups, with carbohydrate, lipid, and xenobiotics metabolism belonging to the EPE and MPE clusters and transcription and translation to the LPE cluster. This study represents a significant advancement in the understanding of the regulation of gene expression by JH and its receptor Met during female mosquito reproduction.

The floral homeotic protein APETALA2 recognizes and acts through an AT-rich sequence element.

Cell fate specification in development requires transcription factors for proper regulation of gene expression. In Arabidopsis, transcription factors encoded by four classes of homeotic genes, A, B, C and E, act in a combinatorial manner to control proper floral organ identity. The A-class gene APETALA2 (AP2) promotes sepal and petal identities in whorls 1 and 2 and restricts the expression of the C-class gene AGAMOUS (AG) from whorls 1 and 2. However, it is unknown how AP2 performs these functions. Unlike the other highly characterized floral homeotic proteins containing MADS domains, AP2 has two DNA-binding domains referred to as the AP2 domains and its DNA recognition sequence is still unknown. Here, we show that the second AP2 domain in AP2 binds a non-canonical AT-rich target sequence, and, using a GUS reporter system, we demonstrate that the presence of this sequence in the AG second intron is important for the restriction of AG expression in vivo. Furthermore, we show that AP2 binds the AG second intron and directly regulates AG expression through this sequence element. Computational analysis reveals that the binding site is highly conserved in the second intron of AG orthologs throughout Brassicaceae. By uncovering a biologically relevant AT-rich target sequence, this work shows that AP2 domains have wide-ranging target specificities and provides a missing link in the mechanisms that underlie flower development. It also sets the foundation for understanding the basis of the broad biological functions of AP2 in Arabidopsis, as well as the divergent biological functions of AP2 orthologs in dicotyledonous plants.

AlignGraph: algorithm for secondary de novo genome assembly guided by closely related references.

MOTIVATION: De novo assemblies of genomes remain one of the most challenging applications in next-generation sequencing. Usually, their results are incomplete and fragmented into hundreds of contigs. Repeats in genomes and sequencing errors are the main reasons for these complications. With the rapidly growing number of sequenced genomes, it is now feasible to improve assemblies by guiding them with genomes from related species. RESULTS: Here we introduce AlignGraph, an algorithm for extending and joining de novo-assembled contigs or scaffolds guided by closely related reference genomes. It aligns paired-end (PE) reads and preassembled contigs or scaffolds to a close reference. From the obtained alignments, it builds a novel data structure, called the PE multipositional de Bruijn graph. The incorporated positional information from the alignments and PE reads allows us to extend the initial assemblies, while avoiding incorrect extensions and early terminations. In our performance tests, AlignGraph was able to substantially improve the contigs and scaffolds from several assemblers. For instance, 28.7-62.3% of the contigs of Arabidopsis thaliana and human could be extended, resulting in improvements of common assembly metrics, such as an increase of the N50 of the extendable contigs by 89.9-94.5% and 80.3-165.8%, respectively. In another test, AlignGraph was able to improve the assembly of a published genome (Arabidopsis strain Landsberg) by increasing the N50 of its extendable scaffolds by 86.6%. These results demonstrate AlignGraph's efficiency in improving genome assemblies by taking advantage of closely related references. AVAILABILITY AND IMPLEMENTATION: The AlignGraph software can be downloaded for free from this site: https://github.com/baoe/AlignGraph.

Arabidopsis lateral organ boundaries negatively regulates brassinosteroid accumulation to limit growth in organ boundaries.

Leaves and flowers begin life as outgrowths from the edges of shoot apical meristems. Stem cell divisions in the meristem center replenish cells that are incorporated into organ primordia at the meristem periphery and leave the meristem. Organ boundaries, regions of limited growth that separate forming organs from the meristem, serve to isolate these two domains and are critical for coordination of organogenesis and meristem maintenance. Boundary formation and maintenance are poorly understood processes, despite the identification of a number of boundary-specific transcription factors. Here we provide genetic and biochemical evidence that the Arabidopsis thaliana transcription factor lateral organ boundaries (LOB) negatively regulates accumulation of the plant steroid hormone brassinosteroid (BR) in organ boundaries. We found that ectopic expression of LOB results in reduced BR responses. We identified BAS1, which encodes a BR-inactivating enzyme, as a direct target of LOB transcriptional activation. Loss-of-function lob mutants exhibit organ fusions, and this phenotype is suppressed by expression of BAS1 under the LOB promoter, indicating that BR hyperaccumulation contributes to the lob mutant phenotype. In addition, LOB expression is BR regulated; therefore, LOB and BR form a feedback loop to modulate local BR accumulation in organ boundaries to limit growth in the boundary domain.

BRANCH: boosting RNA-Seq assemblies with partial or related genomic sequences.

MOTIVATION: De novo transcriptome assemblies of RNA-Seq data are important for genomics applications of unsequenced organisms. Owing to the complexity and often incomplete representation of transcripts in sequencing libraries, the assembly of high-quality transcriptomes can be challenging. However, with the rapidly growing number of sequenced genomes, it is now feasible to improve RNA-Seq assemblies by guiding them with genomic sequences. RESULTS: This study introduces BRANCH, an algorithm designed for improving de novo transcriptome assemblies by using genomic information that can be partial or complete genome sequences from the same or a related organism. Its input includes assembled RNA reads (transfrags), genomic sequences (e.g. contigs) and the RNA reads themselves. It uses a customized version of BLAT to align the transfrags and RNA reads to the genomic sequences. After identifying exons from the alignments, it defines a directed acyclic graph and maps the transfrags to paths on the graph. It then joins and extends the transfrags by applying an algorithm that solves a combinatorial optimization problem, called the Minimum weight Minimum Path Cover with given Paths. In performance tests on real data from Caenorhabditis elegans and Saccharomyces cerevisiae, assisted by genomic contigs from the same species, BRANCH improved the sensitivity and precision of transfrags generated by Velvet/Oases or Trinity by 5.1-56.7% and 0.3-10.5%, respectively. These improvements added 3.8-74.1% complete transcripts and 8.3-3.8% proteins to the initial assembly. Similar improvements were achieved when guiding the BRANCH processing of a transcriptome assembly from a more complex organism (mouse) with genomic sequences from a related species (rat). AVAILABILITY: The BRANCH software can be downloaded for free from this site: http://manuals.bioinformatics.ucr.edu/home/branch. CONTACT: thomas.girke@ucr.edu SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.