WledgmentsWe thank Professor Liu Bin for aid in histological evaluation and George Liu for help of ApoC3 transgenic mice.Author ContributionsConceived and created the experiments: ZY. Performed the experiments: XD YH. Analyzed the data: XD YH HY. Contributed reagents/ materials/analysis tools: XD YH HY. Wrote the paper: XD ZY.
Dinh et al. Silence 2013, four:1 http://silencejournal/content/4/1/METHODSOpen AccessGeneration of a luciferase-based reporter for CHH and CG DNA methylation in Arabidopsis thalianaThanh Theresa Dinh1,2, Michael O’Leary1,4, So Youn Won1, Shengben Li1, Lorena Arroyo1, Xigang Liu1, Andrew Defries1,2, Binglian Zheng1,five, Sean R Cutler1 and Xuemei Chen1,3*AbstractBackground: DNA methylation guarantees genome integrity and regulates gene expression in diverse eukaryotes. In Arabidopsis, methylation occurs in three sequence contexts: CG, CHG and CHH. The initial establishment of DNA methylation at all three sequence contexts occurs by way of a course of action referred to as RNA-directed DNA methylation (RdDM), in which modest RNAs bound by Argonaute4 (AGO4) guide DNA methylation at homologous loci through the de novo methyltransferase DRM2. When established, DNA methylation at every single from the three sequence contexts is maintained through distinct mechanisms. Even though some players involved in RdDM and upkeep methylation happen to be identified, the underlying molecular mechanisms are not completely understood. To help the comprehensive identification of players in DNA methylation, we generated a transgenic reporter program that permits genetic and chemical genetic screens in Arabidopsis. Benefits: A dual 35S promoter (d35S) driven luciferase (LUC) reporter was introduced into Arabidopsis and LUCL, a line with a low basal degree of luciferase activity, was obtained. LUCL was discovered to be a multi-copy, single-insertion transgene that consists of methylated cytosines in CG, CHG and CHH contexts, together with the highest methylation inside the CG context. Methylation was present all through the promoter and LUC coding region. Remedy with an inhibitor of cytosine methylation de-repressed luciferase activity. A mutation in MET1, which encodes the CG maintenance methyltransferase, drastically lowered CG methylation and de-repressed LUC expression. Mutations in AGO4 and DRM2 also de-repressed LUC expression, albeit to a smaller extent than loss of MET1. Working with LUCL as a reporter line, we performed a chemical screen for compounds that de-repress LUC expression, and identified a chemical, methotrexate, identified to be involved in biogenesis on the methyl donor. Conclusion: We developed a luciferase-based reporter system, LUCL, which reports both RdDM and CG upkeep methylation in Arabidopsis.79060-88-1 Data Sheet The low basal degree of LUCL expression provides an easy readout in genetic and chemical genetic screens that may dissect the mechanisms of RdDM and methylation maintenance.Fmoc-Lys(Mtt)-OH structure Keywords and phrases: Luciferase, RdDM, DNA methylation, MET1, AGO4, DRM2, MethotrexateBackground An epigenetic modification that influences gene expression and genome stability is cytosine DNA methylation, which includes the addition of a methyl group to the five position of your pyrimidine cytosine.PMID:33661178 This mark in transposable components or intergenic regions is normally connected with transcriptional gene silencing (TGS) and contributes* Correspondence: [email protected] 1 Department of Botany and Plant Sciences, Institute of Integrative Genome Biology, University of California, Riverside, CA 92521, USA 3 Howard Hughes Medical Inst.
