Arabidopsis circadian clock protein, TOC1, is a DNA-binding transcription factor.
The first described feedback loop of the Arabidopsis circadian clock is based on reciprocal regulation between Timing of CAB Expression 1 (TOC1) and Circadian Clock-associated 1 (CCA1)/late elongated hypocotyl (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 support 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 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 previously unexplored potential TOC1 targets and output pathways. Taken together, these results define a biochemical action for the core clock protein TOC1 and refine our perspective on how plant clocks function.
Section of Cell and Developmental Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, CA 92093-0130, USA.
Joshua M
JM
Gendron
José L
JL
Pruneda-Paz
Colleen J
CJ
Doherty
Andrew M
AM
Gross
S Earl
SE
Kang
Steve A
SA
Kay
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D057906:Circadian Clocks / Q000235:genetics; D018744:DNA, Plant / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D029681:Arabidopsis Proteins; D018744:DNA, Plant; D004268:DNA-Binding Proteins; D012097:Repressor Proteins; C411822:TOC1 protein, Arabidopsis; D014157:Transcription Factors