Project description:We isolated Vascular specific root protoplast using the pWOL:GFP marker line (Birnbaum et al., 2003) and Fluorescence Activated Cell Sorting (FACS). FACS-treated root protoplast from wild type plants (Col0) were used as control. The GFP positive protoplast from the pWOL:GFP line and WT protoplast were used for a ChIP-seq experiment using H3K27me3 and H3K4me3 antibodies.
Project description:We obtained genome-wide digital gene expression tag profiles within the first three days of P. patens protoplast reprogramming. At four time-points during protoplast reprogramming, the transcript levels of 4827 genes changed more than four-fold and their expression correlated with the reprogramming phase. Gene ontology (GO) and pathway enrichment analysis of differentially expressed genes (DEGs) identified a set of significantly enriched GO terms and pathways, most of which were associated with photosynthesis, protein synthesis and stress responses. DEGs were grouped into six clusters that showed specific expression patterns using a K-means clustering algorithm. An investigation of function and expression patterns of genes identified a number of key candidate genes and pathways in early stages of protoplast reprogramming, which provided important clues to reveal the molecular mechanisms responsible for protoplast reprogramming. We identified genes that show highly dynamic changes in expression during protoplast reprogramming into stem cells in P. patens. These genes are potential targets for further functional characterization and should be valuable for exploration of the mechanisms of stem cell reprogramming. In particular, our data provides evidence that protoplasts of P. patens are an ideal model system for elucidation of the molecular mechanisms underlying differentiated plant cell reprogramming. Examination of 4 different sampling times.
Project description:We obtained genome-wide digital gene expression tag profiles within the first three days of P. patens protoplast reprogramming. At four time-points during protoplast reprogramming, the transcript levels of 4827 genes changed more than four-fold and their expression correlated with the reprogramming phase. Gene ontology (GO) and pathway enrichment analysis of differentially expressed genes (DEGs) identified a set of significantly enriched GO terms and pathways, most of which were associated with photosynthesis, protein synthesis and stress responses. DEGs were grouped into six clusters that showed specific expression patterns using a K-means clustering algorithm. An investigation of function and expression patterns of genes identified a number of key candidate genes and pathways in early stages of protoplast reprogramming, which provided important clues to reveal the molecular mechanisms responsible for protoplast reprogramming. We identified genes that show highly dynamic changes in expression during protoplast reprogramming into stem cells in P. patens. These genes are potential targets for further functional characterization and should be valuable for exploration of the mechanisms of stem cell reprogramming. In particular, our data provides evidence that protoplasts of P. patens are an ideal model system for elucidation of the molecular mechanisms underlying differentiated plant cell reprogramming.
Project description:P1 is the major QTL for maysin and chlorogenic acid accumulation in silk. Both compounds were important for plant defenses. Silk is an important reproductive organ that is critical for good seed setting in corn ear and needs to be protected against various stresses, therefore, metabolics compounds (ex: phenolics) were highly enriched in silk. Here we characterize transcriptome changes in maize protoplast, and natural variants of P1 silks, and pericards to characterize the regulatory landscape. Also we evaluated profiles of silk in B73 x A632 hybrids in order to cis and trans specific effect driven by P1 in maize. Our study identifies new P1 targets in the silk and protoplast. Together with the RNA-seq data (P1-rr vs P1-ww in silk and pericarp and protoplast 35S:P1 vs empty vector control), we observed new P1 functions in silk that were not observed in pericarp. Also, Protoplast and silk ChIP-seq in F1 silk, as well as DAP-seq analysis of P1 - shows specific P1 targets with highlight cis and trans effect on the F1 hybrids.
Project description:Autoimmune diseases disproportionately affect females more than males. The XX sex chromosome complement is strongly associated with susceptibility to autoimmunity. Xist long noncoding RNA (lncRNA) is expressed only in females to randomly inactivate one of the two X chromosomes to achieve gene dosage compensation. Here, we show that the Xist ribonucleoprotein (RNP) complex, comprised of numerous autoantigenic components, is an important driver of sex-biased autoimmunity. Inducible transgenic expression of a non-silencing form of Xist in male mice introduced Xist RNP complexes and sufficed to produce autoantibodies. Male SJL/J mice expressing transgenic Xist developed more severe multiorgan pathology in pristane-induced model of lupus than wild-type males. Xist expression in males reprogrammed T and B cell population and chromatin states to more resemble wild type females. Human patients with autoimmune diseases displayed significant autoantibodies to multiple components of XIST RNP. Thus, a sex-specific lncRNA scaffolds ubiquitous RNP components to drive sex-biased immunity.
Project description:C. elegans GLD-2 forms an active PAP with multiple RNA-binding partners to regulate diverse aspects of germline and early embryonic development. One GLD-2 partner, RNP-8, was previously shown to influence oocyte fate specification. To identify transcripts selectively associated with both GLD-2 and RNP-8, we employ a genomic approach using the method of RNA immunoprecipitation followed by microarray analysis (RIP-chip). We used microarrays to identify mRNAs selectively associated with either GLD-2 or RNP-8. Worm extracts were prepared from synchronized adult C. elegans (15 h after L4 stage). For GLD-2 IP, an immoblized anti-GLD-2 antibody was then used to purify the GLD-2 complexes from either wild-type (N2) or gld-2(RNAi) worm extracts. RNA was then extracted from the pellets and analyzed on C.elegans Affymetrix genechip. Four biological replicates were performed, each sample processed in parallel. For RNP-8 IP, an immoblized anti-RNP-8 antibody was then used to purify the RNP-8 complexes from either wild-type (N2) or rnp-8(q784) worm extracts and three biological replicates were performed. For wt or gld-2(RNAi) samples, total RNA was extracted from worm extracts and hybridized on C.elegans Affymetrix genechip.
Project description:RNP granules are membrane-less compartments within cells, formed by phase separation, that function as regulatory hubs for diverse biological processes. However, the mechanisms by which RNAs and proteins interact to promote RNP granule assembly and function in vivo remain unclear. In Xenopus laevis oocytes, maternal mRNAs are transported as large RNPs to the vegetal hemisphere of the developing oocyte, where local translation is critical for proper embryonic patterning. Here, we demonstrate that vegetal transport RNPs represent a new class of cytoplasmic RNP granule, termed Localization-bodies (L-bodies). We show that L-bodies are multiphase RNP granules, containing a dynamic liquid-like protein-containing phase surrounding a non-dynamic RNA-containing substructure. Our results support a role for RNA as a critical scaffold component within these RNP granules and suggest that cis-elements within localized mRNAs may drive subcellular RNA localization through control over phase behavior.
2020-09-20 | GSE158246 | GEO
Project description:protoplast DNA Amplicon sequencing