Proteomic analysis of Drosophila CLOCK complexes identifies rhythmic interactions with SAGA and Tip60 complex component NIPPED-A

Abstract

Circadian clocks keep time via ~ 24 h transcriptional feedback loops. In Drosophila, CLOCK-CYCLE (CLK-CYC) activators and PERIOD-TIMELESS (PER-TIM) repressors are feedback loop components whose transcriptional status varies over a circadian cycle. Although changes in the state of activators and repressors has been characterized, how their status is translated to transcriptional 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 reducing Nipped-A expression lengthens circadian period. RNAi analysis of other SAGA complex components shows that the SAGA histone deubiquitination (DUB) module lengthened period similarly to Nipped-A RNAi 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.

The Clock:Cycle complex is a major transcriptional regulator of Drosophila photoreceptors that protects the eye from retinal degeneration and oxidative stress

“…As a control, we profiled the transcriptome of photoreceptors that expressed LacZ, herein referred as Rh1>Ctrl. We collected both Rh1>Clk DN and Rh1>Ctrl flies at ZT 9, when Clock-dependent transcription is active [48,49], harvesting flies at D1 and D10 to study the progressive effect of disrupting the circadian clock in adult photoreceptors (Fig 3B). We note that Rh1-Gal4 activity begins in the very late stages of pupal development [50]; thus Rh1>Clk DN flies have a disrupted Clk:Cyc complex in adult photoreceptors, but not in the developing eye.…”

Circadian Interactomics: How Research Into Protein-Protein Interactions Beyond the Core Clock Has Influenced the Model of Circadian Timekeeping

“…To elucidate the mechanics that time positive-arm activation and repression, and the protein factors that impart those mechanics, a recent interactomics study focused on identifying proteins in the macromolecular complexes centered on CLK. Over the circadian day, as many as 138 proteins were detected in complex with CLK, with functions ranging from kinases, to phosphatases, to protein degradation machinery components, to chromatin remodelers (Mahesah et al, 2020). Of particular interest, the CLK interactor chromatin remodeler NIPPED-A was found to regulate period length as well as rhythmicity (Murr et al, 2007; Helmlinger and Tora, 2017; Bu et al, 2020; Mahesah et al, 2020).…”

“…Over the circadian day, as many as 138 proteins were detected in complex with CLK, with functions ranging from kinases, to phosphatases, to protein degradation machinery components, to chromatin remodelers (Mahesah et al, 2020). Of particular interest, the CLK interactor chromatin remodeler NIPPED-A was found to regulate period length as well as rhythmicity (Murr et al, 2007; Helmlinger and Tora, 2017; Bu et al, 2020; Mahesah et al, 2020). The interaction and period determining phenotype of NIPPED-A, a scaffold protein in the transcriptional co-activator SAGA/Tip60 complex (Figure 2), suggests a mechanism to facilitate transcriptional activation, establishing an example of the potential importance of macromolecular complexes in the initiation of the circadian cycle.…”

The Clock:cycle complex is a major transcriptional regulator ofDrosophilaphotoreceptors that protects the eye from retinal degeneration and oxidative stress

“…As a control, we profiled the transcriptome of photoreceptors that expressed LacZ, herein referred as Rh1>Ctrl. We collected both Rh1>CLK DN and Rh1>Ctrl flies at ZT 9, when clock-dependent transcription is active (46,47), harvesting flies at D1 and D10 to study the progressive effect of disruption of the circadian clock in adult photoreceptors (Fig. 3B).…”