Proteomic analysis of Drosophila CLOCK complexes identifies rhythmic interactions with SAGA and Tip60 complex component NIPPED-A.
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abstract
Circadian clocks keeptime via~24htranscriptional feedback loops. In Drosophila, CLOCK-CYCLE (CLK-CYC) activators and PERIOD-TIMELESS (PER-TIM) repressors are feedback loop components whose transcriptional status variesoveracircadiancycle. Although changes in the state of activators and repressors has been characterized, how their status istranslated totranscriptional activity is not understood. We used mass spectrometry to identify proteins that interact with GFP-tagged CLK (GFP-CLK) in fly heads at different times of day. Many expected and novel interacting proteins were detected, of which several interacted rhythmically and were potential regulators of protein levels, activity or transcriptional output. Genes encoding these proteins were tested to determine if they altered circadian behavior via RNAi knockdown in clock cells. The NIPPED-A protein, a scaffold for the SAGA and Tip60 histone modifying complexes, interacts with GFP-CLK as transcription is activated, and reducingNipped-Aexpression lengthens circadian period. RNAi analysis of other SAGA complex components shows that the SAGA histone deubiquitination (DUB) module lengthened period similarly toNipped-ARNAi knockdown and weakened rhythmicity, whereas reducing Tip60 HAT expression drastically weakened rhythmicity. These results suggest that CLK-CYC binds NIPPED-A early in the day to promote transcription through SAGA DUB and Tip60 HAT activity.
