Project description:Pitx2 is the homeobox gene located in proximity to the human 4q25 familial atrial fibrillation locus. Pitx2 haploinsufficient mice are prone to pacing induced atrial fibrillation indicating that reduced Pitx2 promotes an arrhythmogenic substrate within the atrium. Here, we inactivated Pitx2 in postnatal heart and discovered that unstressed adult Pitx2 mutant mice had sinus node dysfunction with impaired atrial conduction, an arrhythmia closely associated with atrial fibrillation. A genome-wide search for Pitx2 transcriptional targets using ChIP-sequencing and RNA expression profiling shows that Pitx2 represses target genes encoding cell junction proteins, ion channels, and critical transcriptional regulators many of which have been implicated in human atrial fibrillation by genome wide association studies. Our findings unveil a Pitx2 postnatal arrhythmogenic function, novel Pitx2 target genes relevant to atrial fibrillation, and reveal that Pitx2 stabilizes the intercalated disc in postnatal atrium. Genomic occupancy profiling of transcriptional factor Pitx2 in postnatal heart.
Project description:ChIP-seq analysis of HepG2 cells revealed that many of the target genes of LSD2 were related to lipid metabolism. We found that LSD2 is an important epigenetic regulator of hepatic lipid metabolism. Examination of LSD2/DNA interaction in HepG2 cells in normal condition.
Project description:ChIP-seq analysis of LSD2-depleted HepG2 cells revealed that many of the target genes were related to lipid metabolism. We found that LSD2 is an important epigenetic regulator of hepatic lipid metabolism. Examination of LSD2/H3K4me1 interaction in control and LSD2-knockdowned HepG2 cells.
Project description:Pitx2 is the homeobox gene located in proximity to the human 4q25 familial atrial fibrillation locus. Pitx2 haploinsufficient mice are prone to pacing induced atrial fibrillation indicating that reduced Pitx2 promotes an arrhythmogenic substrate within the atrium. Here, we inactivated Pitx2 in postnatal heart and discovered that unstressed adult Pitx2 mutant mice had sinus node dysfunction with impaired atrial conduction, an arrhythmia closely associated with atrial fibrillation. A genome-wide search for Pitx2 transcriptional targets using ChIP-sequencing and RNA expression profiling shows that Pitx2 represses target genes encoding cell junction proteins, ion channels, and critical transcriptional regulators many of which have been implicated in human atrial fibrillation by genome wide association studies. Pitx2 control and mutant hearts were collected from 3-, 6- and 12-week-old mice. At each time point, three cotrols and three mutants were collected as biological replicates. cDNA microarray analysis was performed using Affymetrix GeneChip Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara, CA).
Project description:LHY and CCA1 encode single MYB transcription factors, involved in circadian clock. However, direct target genes of LHY and CCA1 in a genomic scale were largely unknown. To reveal bound genes by CCA1, chimeric protein CCA1-FLAG was expressed under CCA1 promoter in cca1 lhy (CCA1pro:CCA1-FLAG/ cca1 lhy). ChIP was performed using anti-FLAG antibody (F3165; SIGMA), which was bound to Dynabeads Protein G (100-03D; Life Technologies), and ChIP DNA were analyzed by IonPGM or Illumina GAII. Chromatin immunoprecipitation was performed for CCA1-FLAG-expressing Arabidopsis. ChIP DNA was analyzed 2 types of deep sequencers (Illumina GAII and IonPGM).
Project description:Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) is a widely used approach to study DNA methylation genome-wide. Here, we present a novel MeDIP-Seq protocol compatible with the Ion Torrent semiconductor-based sequencing platform that is scalable and accurately identifies sites of differential DNA methylation. Additionally, we demonstrate that the high-throughput data derived from MeDIP-Seq on the Ion Torrent platform provides adequate coverage of CpG cytosines, the methylation states of which we validated at single-base resolution on the Infinium HumanMethylation450K Beadchip array. We applied this integrative approach to further investigate the role of DNA methylation in alternative splicing and to profile 5-mC and 5-hmC variants of DNA methylation in normal human brain tissue that we observed localize over distinct genomic regions. These applications of MeDIP-Seq on the Ion Torrent platform have broad utility and add to the current methodologies for profiling genome-wide DNA methylation states in normal and disease conditions. MeDIP-Seq on Ion Torrent Platform in HCT116 and Human Brain
Project description:Background: Microorganisms are the major cause of food spoilage during storage, processing and distribution. Pseudomonas fluorescens is a typical spoilage bacterium that contributes to a large extent to the spoilage process of proteinaceous food. RpoS is considered an important global regulator involved in stress survival and virulence in many pathogens. Our previous work revealed that RpoS contributed to the spoilage activities of P. fluorescens by regulating resistance to different stress conditions, extracellular acylated homoserine lactone (AHL) levels, extracellular protease and total volatile basic nitrogen (TVB-N) production. However, RpoS-dependent genes in P. fluorescens remained undefined. Results: RNA-seq transcriptomics analysis combined with quantitative proteomics analysis basing on multiplexed isobaric tandem mass tag (TMT) labeling was performed for the P. fluorescens wild-type strain UK4 and its derivative carrying a rpoS mutation. A total of 375 differentially expressed genes (DEGs) and 212 differentially expressed proteins (DEPs) were identified in these two backgrounds. The DGEs were further verified by qRT-PCR tests, and the genes directly regulated by RpoS were confirmed by 5’-RACE-PCR sequencing. The combining transcriptome and proteome analysis revealed a role of this regulator in several cellular processes, including polysaccharide metabolism, intracellular secretion and extracellular structures, cell well biogenesis, stress responses, ammonia and biogenic amine production, which may contribute to biofilm formation, stress resistance and spoilage activities of P. fluorescens. Moreover, in this work we indeed observed that RpoS contributed to the production of the macrocolony biofilm’s matrix.
Project description:Single-cell expression profiling by RNA-Seq promises to exploit cell-to-cell variation in gene expression to reveal regulatory circuitry governing cell differentiation and other biological processes. Here, we describe Monocle, a novel unsupervised algorithm for ordering cells by progress through differentiation that dramatically increases temporal resolution of expression measurements. This reordering unmasks switch-like changes in expression of key regulatory factors, reveals sequentially organized waves of gene regulation, and exposes regulators of cell differentiation. A functional screen confirms that a number of these regulators dramatically alter the efficiency of myoblast differentiation, demonstrating that single-cell expression analysis with Monocle can uncover new regulators even in well-studied systems. We selected primary human myoblasts as a model system of cell differentiation to investigate whether ordering cells by progress revealed new regulators of the process. We sequenced RNA-Seq libraries from each of several hundred cells taken over a time-course of serum-induced differentiation. Please note that this dataset is a single-cell RNA-Seq data set, and each cell comes from a capture plate. Thus, each well of the plate was scored and flagged with several QC criteria prior to library construction, which are provided as sample characteristics; CONTROL indicates that this library is a off-chip tube control library constructed from RNA of approximately 250 cells and 'DEBRIS' indicates that the well contained visible debris (and may or may not include a cell). Libraries marked DEBRIS thus cannot be confirmed to come from a single cell.