We show that diminished levels or activity of the TPL family causes increased levels of CCA1 and LHY expression, and a concomitant lengthening of circadian period. Here we identify members of the plant Groucho/TUP1 corepressor family, TOPLESS/TOPLESS RELATED PROTEINs (TPL/TPRs), which specifically interact with three of the five members of the PRR family (PRR5, PRR7, and PRR9) and reside together at the promoters of CCA1 and LHY to repress transcription and alter the circadian period. However, the mechanism of how these proteins inhibit expression remains unknown. These results, together with the recent demonstration of DNA binding by these PRRs ( 8, 17), provide compelling evidence that PRR9, PRR7, and PRR5 act in temporal sequence to keep CCA1 and LHY transcription strongly repressed over most of the midmorning to early evening. Mutants lacking two of the three PRR proteins often display altered patterns of CCA1 and LHY expression, with increased expression of both genes coinciding with circadian times at which the missing PRRs would normally be expressed ( 15). These protein expression patterns closely mirror their temporal occupancy of CCA1 and LHY promoter regions ( 15). PRR7 peaks next between ZT6 and ZT13 and PRR5 follows near ZT13 ( 15, 16). PRR9 accumulation begins early in the day, with maximum levels found between zeitgeber time (ZT) 2–6. Each of these PRRs is expressed at discrete times of the circadian cycle. In addition to the role of TOC1, establishment and regulation of CCA1 and LHY circadian expression relies on repression by three additional PRRs, PRR9, PRR7, and PRR5 ( 15). However, a second evening-phased repressor complex, EARLY FLOWERING 3 (ELF3)-EARLY FLOWERING4 (ELF4)-LUX ARRHYTHMO (LUX) has been identified as acting to restrict PRR9 expression to the morning ( 12 – 14). In contrast, the partners and mechanism of late-evening TOC1-mediated repression of CCA1/LHY are unknown. TOC1 is also regulated by the related myb-transcription factor, REV8, which binds the TOC1 promoter and likely acts as a positive activator ( 10, 11). The mechanism of CCA1/LHY-mediated repression of TOC1 requires the corepressor DE-ETIOLATED1 (DET1) to interact with CCA1 and LHY at the TOC1promoter, likely in the context of a larger COP10-DET1-DDB1(CDD) complex ( 9). TOC1 is the founding member of five closely related PSEUDO RESPONSE REGULATORS (PRRs: PRR9, PRR7, PRR5, PRR3) and binds DNA through a conserved CCT domain at the carboxy terminus, repressing evening expression of both CCA1 and LHY ( 7, 8). One well-studied loop of reciprocal repression involves the inhibition of early-day expression of the evening gene TIMING OF CAB EXPRESSION 1 (TOC1 PRR1) by the morning-expressed myb transcription factors CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) ( 6). Many of the best-characterized elements in the plant circadian system are transcriptional repressors that act during the subjective morning to allow evening expression of their targets, or are expressed during the subjective evening to keep expression of morning genes down at night ( 4, 5). The circadian clock system consists of multiple interlocked feedback loops that generally contain activating and repressive arms within the loops to sustain robust 24-h oscillations ( 1 – 3). Our findings show that the TPL/TPR corepressor family are components of the central circadian oscillator mechanism and reinforces the role of this family as central to multiple signaling pathways in higher plants. Additionally, a complex of PRR9, TPL, and histone deacetylase 6, can form in vivo, implicating this tripartite association as a central repressor of circadian gene expression. This activity is diminished in the presence of the inhibitor trichostatin A, indicating the requirement of histone deacetylase for full TPL activity. Here we report that members of the plant Groucho/Tup1 corepressor family, TOPLESS/TOPLESS-RELATED (TPL/TPR), interact with these three PRR proteins at the CCA1 and LHY promoters to repress transcription and alter circadian period. PRR5, PRR7, and PRR9 can bind the promoters of the core clock genes CIRCADIAN CLOCK ASSOCIATED 1 ( CCA1) and LATE ELONGATED HYPOCOTYL ( LHY) to restrict their expression to near dawn, but the mechanism has been unclear. PSEUDO RESPONSE REGULATORS (PRRs) comprise a five-member family that is essential to the function of the central oscillator. The central oscillator of the plant clock is composed of interlocked feedback loops that involve multiple repressive factors acting throughout the circadian cycle. Circadian clocks are ubiquitous molecular time-keeping mechanisms that coordinate physiology and metabolism and provide an adaptive advantage to higher plants.
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