Project description:We have examined the roles of yeast mRNA decapping-activators Pat1 and Dhh1 in repressing the translation and abundance of specific mRNAs in nutrient-replete cells using ribosome profiling, RNA-Seq, CAGE analysis of capped mRNAs, RNA Polymerase II ChIP-Seq, and TMT-mass spectrometry of mutants lacking one or both factors. Although the Environmental Stress Response (ESR) is activated in dhh1 and pat1 mutants, hundreds of non-ESR transcripts are elevated in a manner indicating cumulative repression by Pat1 and Dhh1 in WT. These mRNAs show reduced decapping and diminished transcription in the mutants, indicating that impaired mRNA turnover drives transcript derepression in cells lacking Dhh1 or Pat1. mRNA degradation stimulated by Dhh1/Pat1 is not dictated by poor translation nor enrichment for suboptimal codons. Pat1 and Dhh1 also collaborate to reduce translation and protein production from many mRNAs. Transcripts showing concerted translational repression by Pat1/Dhh1 include mRNAs involved in cell adhesion or utilization of the poor nitrogen source allantoin. Pat1/Dhh1 also repress numerous transcripts involved in respiration, catabolism of non-preferred carbon or nitrogen sources, or autophagy; and we obtained evidence for elevated respiration and autophagy in the mutants. Thus, Pat1 and Dhh1 function as post-transcriptional repressors of multiple pathways normally activated only during nutrient limitation.
Project description:We identified and characterized that the rice Dioxygenase for Auxin Oxidation (dao) mutant displays pleiotropic phenotypes, including indehiscent anthers, sterile pollens, and increased free IAA levels in anthers.We conclude that DAO-mediated auxin catabolism is essential for auxin homeostasis and later stages of plant reproductive development, including anther dehiscence, and pollen viability. We used microarrays to identify differentially expressed genes in dao.
Project description:Sphingolipids are required for diverse biological functions and are degraded by specific catabolic enzymes. However, the mechanisms that regulate sphingolipid catabolism are not known. Here we characterize a transcriptional axis that regulates sphingolipid breakdown to control resistance against bacterial infection. From an RNAi screen for transcriptional regulators of pathogen resistance in the nematode C. elegans, we identified the nuclear hormone receptor nhr-66, a ligand-gated transcription factor homologous to human hepatocyte nuclear factor 4. Tandem chromatin immunoprecipitation-sequencing (ChIP-seq) and RNA sequencing (RNA-seq) experiments revealed that NHR-66 is a transcriptional repressor, which directly targets sphingolipid catabolism genes. Transcriptional de-repression of two sphingolipid catabolic enzymes in nhr-66 loss-of-function mutants drives the breakdown of sphingolipids, which enhances host susceptibility to infection with the bacterial pathogen Pseudomonas aeruginosa. These data define transcriptional control of sphingolipid catabolism in the regulation of cellular sphingolipids, a process that is necessary for pathogen resistance.
Project description:KNOTTED1(KN1)-like homeobox (KNOX) transcription factors function in plant meristems, self-renewing structures consisting of stem cells and their immediate daughters. Despite their importance for plant development, the genomic network targeted by KNOX proteins is poorly understood. Using ChIP-seq, we defined the KN1 cistrome in maize inflorescences and found that KN1 binds to several thousand loci. To understand how these binding occupancies correlate with changes in transcriptional regulation, we performed RNA-seq on immature ears and tassels, and compared expression profiles between normal and loss-of-function kn1 plants, in addition to immature leaves from normal and gain-of-function Kn1 plants. We found that 643 of the KN1 targets were modulated in one or multiple tissues, with a strong enrichment for transcription factors (including other homeobox genes) and genes participating in several hormonal pathways, most significantly auxin, implicating KN1 at the crossroads of plant hormone signaling. The loss-of-function kn1 phenotype is reminiscent of auxin mutants and kn1 mis-expression in leaves correlates with increased auxin signaling. Our results demonstrate that KN1 plays a key role in orchestrating the upper levels of a hierarchical gene regulatory network that impacts plant meristem identity and function. ChIP-seq was performed using ear primordia and tassel primordia. Input DNA from each sample was used as a normalization control
Project description:We identified and characterized that the rice Dioxygenase for Auxin Oxidation (dao) mutant displays pleiotropic phenotypes, including indehiscent anthers, sterile pollens, and increased free IAA levels in anthers.We conclude that DAO-mediated auxin catabolism is essential for auxin homeostasis and later stages of plant reproductive development, including anther dehiscence, and pollen viability. We used microarrays to identify differentially expressed genes in dao. For genome-wide expression analysis of dao, three replicates of WT and dao samples (RNA from mature anthers) were analyzed on Affymetrix Genechip® Rice Genome arrays by an Affymetrix service facility (CapitalBio Corporation) according to the manufacturer’s protocols. Genes showing a 2-fold change with a q-value ≤ 0.05 were considered to be differentially expressed.
Project description:We have examined the roles of yeast mRNA decapping-activators Pat1 and Dhh1 in repressing the translation and abundance of specific mRNAs in nutrient-replete cells using a combination of ribosome profiling, RNA-Seq, CAGE analysis of capped mRNAs, RNA Polymerase II ChIP-Seq, and TMT-mass spectrometry of mutants lacking one or both factors.
Project description:Auxin is a major plant hormone for both development and environmental adaptation. Auxin responses are context dependent and highly modulated by light, temperature, the circadian clock, brassinosteroid, and gibberellin, but the underlying mechanisms remain unclear. Here, we show that auxin signaling integrates with other signals through direct interactions of AUXIN RESPONSE FACTOR6 (ARF6) with PHYTOCHROME INTERACTING FACTOR4 (PIF4), the brassinosteroid-signaling transcription factor BZR1, and the gibberellin-signaling repressor RGA. ChIP-Seq and RNA-Seq experiments show that ARF6, PIF4, and BZR1 bind to largely overlapping targets in the genome and synergistically activate gene expression. In vitro and in vivo assays show that ARF6-promoter binding is enhanced by PIF4 and BZR1 but blocked by RGA. Furthermore, a tripartite HLH/bHLH module feedback regulates PIF activity and thus modulates auxin sensitivity according to additional developmental and environmental cues. Our results demonstrate a central growth-regulation transcriptional network that coordinates hormonal, environmental, and developmental control of cell elongation and plant growth. Genome-wide identification of ARF6 DNA-binding sites in etiolated Arabidopsis seedlings.
Project description:To study the role of the exonuclease Xrn1 in translational control, we performed ribosome profiling and RNA-seq in Xrn1-depleted cells. By using an auxin-inducible degron, we were able to study immediate effects of Xrn1 depletion in translational control. Therefore, we could overcome experimental limitations associated to stable deletion mutants.
Project description:To study the role of the exonuclease Xrn1 in gene expression dynamics under osmotic stress conditions, we performed RNA-seq in Xrn1-depleted cells. By using an auxin-inducible degron, we were able to study immediate effects of Xrn1 depletion in gene expression dynamics. Therefore, we could overcome experimental limitations associated to stable deletion mutants.