dCLOCK is present in limiting amounts and likely mediates daily interactions between the dCLOCK-CYC transcription factor and the PER-TIM complex

Abstract

In Drosophila melanogaster four circadian clock proteins termed PERIOD (PER), TIMELESS (TIM), dCLOCK (dCLK), and CYCLE (CYC/dBMAL1) function in a transcriptional feedback loop that is a core element of the oscillator mechanism. dCLK and CYC are members of the basic helix-loop-helix (bHLH)/PAS (PER-ARNT-SIM) superfamily of transcription factors and are required for high-level expression of per and tim and repression of dClk, whereas PER and TIM inhibit dCLK-CYC-mediated transcription and lead to the activation of dClk. To understand further the dynamic regulation within the circadian oscillator mechanism, we biochemically characterized in vivo-produced CYC, determined the interactions of the four clock proteins, and calculated their absolute levels as a function of time. Our results indicate that throughout a daily cycle the majority of the dCLK present in adult heads stably interacts with CYC, indicating that CYC is the primary in vivo partner of dCLK. dCLK-CYC dimers are bound by PER and TIM during the late evening and early morning, suggesting the formation of a tetrameric complex with impaired transcriptional activity. Although dCLK is present in limiting amounts and CYC is by far the most abundant of the four clock proteins that have been examined, PER and TIM appear to interact preferentially with dCLK. Our results suggest that dCLK is the main component regulating the daily abundance of transcriptionally active dCLK-CYC complexes.

Fig. 1.

Biochemical detection of CYC in Drosophila melanogaster heads. Cell-free extracts were derived from eitherin vitro translation reactions performed in the presence of [³⁵S]methionine (A,B,lanes1–4, andC) or adult fly heads (A,B,lanes5, 6). The radiolabeled target proteins produced in vitro (i.e., DBT, PER, TIM, dCLK, and CYC) and the genotype of flies that were used to prepare head extracts (i.e., wild-type CS flies orcyc⁰ mutants) are indicated ontop. Head extracts were prepared from flies collected at time 25 (in hours since the last dark/light transition at ZT0).C, In vitro translation products were incubated with the indicated antibodies, and immune complexes were recovered. Fly head extracts, in vitro translation reactions, and immune complexes were resolved by 6 or 12% (in the case of CYC) PAGE and either visualized by fluorography and autoradiography (A, C) or transferred to nitrocellulose; the immunoblots were probed with anti-CYC antibodies (B).A, B, Two different amounts of in vitro-translated CYC were resolved by PAGE (1× = 77 pg).B, The arrow (left) and asterisk (right) identify nonspecific bands that cross-react with the anti-CYC antibody in rabbit reticulocyte lysates (lanes1–4) and head extracts (lanes5, 6), respectively.

Fig. 2.

Constitutive levels of CYC protein and RNA throughout a daily cycle. Wild-type flies were exposed to three 12 hr light/dark cycles (LD) and subsequently were kept in constant dark conditions (DD). Collections were done at the indicated times (time 0 is defined as the last dark-to-light transition), and head extracts either were analyzed by immunoblotting, using antibodies directed against CYC (A), or were subjected to RNase protection assays (B). A, As a control for specificity, head extracts prepared fromcyc⁰ flies were included (lane 13). B, Comparison of the relative amounts ofcyc (open diamond) and per(open square) RNA during the third day of LD and the first day of DD. Relative RNA levels refers to ratios ofcyc or per transcripts to the constitutively expressed RP49 RNA. Peak values for cycor per during a daily cycle were set to 100, and the rest of the values were normalized. Horizontal barsrepresent lights-on (open bar), lights-off (filled bar), or subjective day (hatched bar). Similar results were obtained in three independent experiments; representative examples are shown.

Fig. 3.

dCLK, PER, and TIM interact with CYC in a time-of-day specific manner. Wild-type (CS) flies were collected at the indicated times in LD (A, lanes1–7; B, lanes1–6). per⁰¹(A, lane 8; B, lane 7) and tim⁰(A, lane 9; B, lane 8) mutants were collected at time 23.5. Head extracts were prepared and either subjected to immunoprecipitation using antibodies against CYC (A) or analyzed directly (B). A, Immune pellets were divided into three equal aliquots; each fraction was probed for the presence of dCLK (top), PER (middle), or TIM (bottom). A,B, Twelve percent polyacrylamide gels were used to detect CYC, whereas 6% polyacrylamide gels were used to detect PER, TIM, and dCLK. The size ranges of the relevant proteins are indicated (left). The arrow (left, top panelin B) indicates a nonspecific band recognized by the anti-dCLK antibody that was used. C,D, Quantitation of results shown in A and B, respectively. Peak values for each protein were set to 100, and the rest of the values were normalized. Horizontal barsrepresent either 12 hr light (open bar) or 12 hr dark (filledbar).

Fig. 4.

Average daily levels of dCLK are limiting. Shown are the molar concentrations (10⁻¹⁸ mol/μg total head protein) of dCLK, CYC, PER, and TIM during LD. For each protein the peak amounts were calculated by pooling results obtained from at least two independent experiments. The rest of the data points were generated by curve fitting, using the results shown in Figure3D (see Materials and Methods). Peak concentrations (10⁻¹⁸ mol/μg total head protein) for each protein also are indicated. Note that, because PER has a very broad electrophoretic mobility during the late night/early morning attributable to differential phosphorylation (Edery et al., 1994), we believe that PER levels during these times might be overestimated by up to 50% (data not shown).

Fig. 5.

The majority of dCLK is bound to CYC during a daily cycle. A,B, Wild-type flies were collected at the indicated times during LD; head extracts were prepared and subjected to immunoprecipitation (IP), using antibodies against either CYC (A, lanes1–4, 7, 8) or dCLK (A, lane 6;B, lanes2–7). In some cases the remaining supernatant fraction subsequently was subjected to a second round of IP, using antibodies against either dCLK (A, lanes2, 4, 8) or CYC (B, lanes3, 4, 6, 7). Finally, in some cases the supernatant resulting from the second IP was subjected to a third round of IP, using antibodies against PER (B, lanes4, 7). Recovered immune complexes were probed for the presence of dCLK, PER, or TIM as indicated (left of panels). Control incubations using irrelevant antibodies were used to show specificity during IP (A, lane 5; B, lane 1) (data not shown).