Project description:Background: There is a growing interest in the role of chromatin in acquiring and maintaining cell identity. Despite the ever growing availability of genome-wide gene expression data, understanding how transcription programs are established and regulated to define cell identity remains a puzzle. An important mechanism of gene regulation is the binding of transcription factors to specific DNA sequence motifs across the genome. However, these sequences are hindered by the packaging of DNA to chromatin. Thus the accessibility of these loci for TF binding is highly regulated and determines where and when TF bind. We present a workflow for measuring chromatin accessibility in Arabidopsis thaliana, and define organ-specific regulatory sites and binding motifs of transcription factors at these sites. Results: We coupled the recently described INTACT (Isolation of Nuclei TAgged in specific Cell Types) and ATAC-seq (Assay for Transposase-Accessible Chromatin with highthroughput sequencing) as a genome-wide strategy to uncover accessible regulatory sites in Arabidopsis, based on their accessibility to nuclease digestion. By applying this pipeline in Arabidopsis roots, we revealed 41,419 accessible sites, of which approximately half are found in gene promoters and contain the H3K4me3 active histone mark. The root-unique accessible sites from this group are enriched for root processes. Interestingly, most of the root-unique accessible sites are found in nongenic regions, but are correlated with root-specific expression of distant genes. Importantly these gene-distant sites are enriched for binding motifs of TFs important for root development, as well as motifs for TFs that may play a role as novel transcriptional regulators in roots, suggesting that these accessible loci are functional novel genedistant regulatory elements. Conclusion: By coupling the INTACT with ATAC-seq methods, we present a feasible pipeline to profile accessible chromatin in plants. We also introduce a rapid measure of the experiment quality. We find that chromatin accessibility at promoter regions is strongly associated with transcription and active histone marks. However, root specific chromatin accessibility is primarily found at intergenic regions, suggesting their predominance in defining organ identity, possibly via long-range chromatin interactions. This workflow can be rapidly applied to study the regulatory landscape in other cell types, plant species and conditions.
Project description:Background: There is a growing interest in the role of chromatin in acquiring and maintaining cell identity. Despite the ever growing availability of genome-wide gene expression data, understanding how transcription programs are established and regulated to define cell identity remains a puzzle. An important mechanism of gene regulation is the binding of transcription factors to specific DNA sequence motifs across the genome. However, these sequences are hindered by the packaging of DNA to chromatin. Thus the accessibility of these loci for TF binding is highly regulated and determines where and when TF bind. We present a workflow for measuring chromatin accessibility in Arabidopsis thaliana, and define organ-specific regulatory sites and binding motifs of transcription factors at these sites. Results: We coupled the recently described INTACT (Isolation of Nuclei TAgged in specific Cell Types) and ATAC-seq (Assay for Transposase-Accessible Chromatin with highthroughput sequencing) as a genome-wide strategy to uncover accessible regulatory sites in Arabidopsis, based on their accessibility to nuclease digestion. By applying this pipeline in Arabidopsis roots, we revealed 41,419 accessible sites, of which approximately half are found in gene promoters and contain the H3K4me3 active histone mark. The root-unique accessible sites from this group are enriched for root processes. Interestingly, most of the root-unique accessible sites are found in nongenic regions, but are correlated with root-specific expression of distant genes. Importantly these gene-distant sites are enriched for binding motifs of TFs important for root development, as well as motifs for TFs that may play a role as novel transcriptional regulators in roots, suggesting that these accessible loci are functional novel genedistant regulatory elements. Conclusion: By coupling the INTACT with ATAC-seq methods, we present a feasible pipeline to profile accessible chromatin in plants. We also introduce a rapid measure of the experiment quality. We find that chromatin accessibility at promoter regions is strongly associated with transcription and active histone marks. However, root specific chromatin accessibility is primarily found at intergenic regions, suggesting their predominance in defining organ identity, possibly via long-range chromatin interactions. This workflow can be rapidly applied to study the regulatory landscape in other cell types, plant species and conditions.
Project description:Transcript profiling analysis of Hydraulic conductivity of Root 1 (HCR1) mutant compared to wild type (Col-0) using ARABIDOPSIS GENE1.1ST ARRAY STRIP (901793, Affymetrix, Santa Clara, USA).
Project description:Col-0 floral stem was grafted on the msh1 mutant (Col-0/msh1); on the dcl2,3,4,msh1 quadruple mutant (Col-0/dcl2,3,4,msh1); on Col-0 (Col-0/Col-0). Seeds were collected from the grafted Col-0 scion after grafts were established. Seed coming from the graft then were grown on the peat mix, leaf tissue was collected at the bolting and used for the total RNA sequencing.
Project description:Ca2+ signaling is central to plant development, modulating gene expression to enable highly specific plant responses. While Ca2+-responsive proteins have been investigated intensely in plants, only few Ca2+ channels are known, and our understanding of how intracellular Ca2+ fluxes are facilitated remains limited. We obtained an Arabidopsis triple knockout mutant of homologues of the mammalian mitochondrial Ca2+ channel-forming MCU protein, in which mitochondrial Ca2+ uptake was severly perturbed in vivo in roots. To pinpoint the impact of the distrupted mitochondrial matrix free Ca2+ dynamcis, we performed transcriptome analysis in roots of an MCU triple knockout background (mcu123) and its corresponding wild type background (Col-0). We devised a microarray-based analysis to investigate transcriptome differences in roots of mcu123 triple knockout backgroudn and Col-0, without additional stimulation, and idenfitied distinct classes of differentially abundant transcripts.
Project description:To investigate the deposition of HTR5 in Arabidopsis, we analysed genome-wide HTR5 density in the wild-type Col-0 by ChIP-seq. We then performed HTR5 occupancy analysis using data obtained from ChIP-seq of 3 different plants including HA-HTR5/Col-0 and Col-0. Col-0 acted as negative control.
Project description:Col-0 floral stem was grafted on the msh1 mutant (Col-0/msh1); on the dcl2,3,4,msh1 quadruple mutant (Col-0/dcl2,3,4,msh1); on Col-0 (Col-0/Col-0). Seeds were collected from the grafted Col-0 scion after grafts were established. Seed coming from the graft then were grown on the peat mix, leaf tissue was collected at the bolting and used for the bisulfite sequencing (methylome). Tissue from the msh1 mutant and dcl2,3,4,msh1 quadruple mutants used as rootstocks was similarly collected at the bolting stage and used for the bisulfite sequencing.