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.
Project description:The first described feedback loop of the Arabidopsis circadian clock is based on reciprocal regulation between TOC1 and CCA1/LHY. CCA1 and LHY are MYB transcription factors that bind directly to the TOC1 promoter to negatively regulate its expression. Conversely, the activity of TOC1 has remained less well characterized. Genetic data supports that TOC1 is necessary for the reactivation of CCA1/LHY, but there is little description of its biochemical function. Here we show that TOC1 occupies specific genomic regions in the CCA1 and LHY promoters. Purified TOC1 binds directly to DNA through its CCT domain, which is similar to known DNA binding domains. Chemical induction and transient overexpression of TOC1 in Arabidopsis seedlings cause repression of CCA1/LHY expression demonstrating that TOC1 can repress direct targets, and mutation or deletion of the CCT domain prevents this repression showing that DNA binding is necessary for TOC1 action. Furthermore, we use the Gal4/UAS system in Arabidopsis to show that TOC1 acts as a general transcriptional repressor, and that repression activity is in the Pseudoreceiver (PR) domain of the protein. To identify the genes regulated by TOC1 on a genomic scale, we couple TOC1 chemical induction with microarray analysis and identify new potential TOC1 targets and output pathways. Together these results define the biochemical action of the core clock protein TOC1 and refine our perspective on how plant clocks function. Keywords: Expression profiling by array
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:The first described feedback loop of the Arabidopsis circadian clock is based on reciprocal regulation between TOC1 and CCA1/LHY. CCA1 and LHY are MYB transcription factors that bind directly to the TOC1 promoter to negatively regulate its expression. Conversely, the activity of TOC1 has remained less well characterized. Genetic data supports that TOC1 is necessary for the reactivation of CCA1/LHY, but there is little description of its biochemical function. Here we show that TOC1 occupies specific genomic regions in the CCA1 and LHY promoters. Purified TOC1 binds directly to DNA through its CCT domain, which is similar to known DNA binding domains. Chemical induction and transient overexpression of TOC1 in Arabidopsis seedlings cause repression of CCA1/LHY expression demonstrating that TOC1 can repress direct targets, and mutation or deletion of the CCT domain prevents this repression showing that DNA binding is necessary for TOC1 action. Furthermore, we use the Gal4/UAS system in Arabidopsis to show that TOC1 acts as a general transcriptional repressor, and that repression activity is in the Pseudoreceiver (PR) domain of the protein. To identify the genes regulated by TOC1 on a genomic scale, we couple TOC1 chemical induction with microarray analysis and identify new potential TOC1 targets and output pathways. Together these results define the biochemical action of the core clock protein TOC1 and refine our perspective on how plant clocks function. Keywords: Expression profiling by array wild type (Col-0) and ALC::TOC1 were sown on Murashige-Skoog with 0.8% agar, stratified for 48 hours and grown in12:12 light:dark (LD) for 12 days and either left in LD or transferred to constant light (LL) and then grown for one more day before the start of the experiment. Tissue was submerged in Murashige-Skoog media supplemented with 2.5% ethanol or no ethanol (mock) and with 20mM MG132 for 3 hours and harvested at ZT1. Three replicates each of the seedlings were collected and frozen in liquid nitrogen.