PER-dependent rhythms in CLK phosphorylation and E-box binding regulate circadian transcription - PubMed (original) (raw)

PER-dependent rhythms in CLK phosphorylation and E-box binding regulate circadian transcription

Wangjie Yu et al. Genes Dev. 2006.

Abstract

Transcriptional activation by CLOCK-CYCLE (CLK-CYC) heterodimers and repression by PERIOD-TIMELESS (PER-TIM) heterodimers are essential for circadian oscillator function in Drosophila. PER-TIM was previously found to interact with CLK-CYC to repress transcription, and here we show that this interaction inhibits binding of CLK-CYC to E-box regulatory elements in vivo. Coincident with the interaction between PER-TIM and CLK-CYC is the hyperphosphorylation of CLK. This hyperphosphorylation occurs in parallel with the PER-dependent entry of DOUBLE-TIME (DBT) kinase into a complex with CLK-CYC, where DBT destabilizes both CLK and PER. Once PER and CLK are degraded, a novel hypophosphorylated form of CLK accumulates in parallel with E-box binding and transcriptional activation. These studies suggest that PER-dependent rhythms in CLK phosphorylation control rhythms in E-box-dependent transcription and CLK stability, thus linking PER and CLK function during the circadian cycle and distinguishing the transcriptional feedback mechanism in flies from that in mammals.

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Figures

Figure 1.

CLK-CYC heterodimers rhythmically bind E-boxes in vivo. (A) ChIP assays were performed on wild-type flies collected at the indicated times during LD cycles. Relative levels of the per CRS E-box present in each chromatin extract (Input) and immunoprecipitates from these chromatin extracts using CLK antibody (CLK), CYC antibody (CYC), and control Guinea pig serum (GPS) are seen as per CRS E-box containing PCR amplification products. (B) ChIP assays were performed as described in A, except that relative levels of the tim upstream E-box (tim UP E-box) are shown. (C) Quantification of per CRS ChIP assays. Error bars represent the range from two independent experiments. The gray line represents per transcription derived from nuclear run-on experiments (So and Rosbash 1997). (D) Quantification of tim UP E-box ChIP assays. Error bars represent the range from two independent experiments. The gray line represents tim transcription derived from nuclear run-on experiments (So and Rosbash 1997).

Figure 2.

Rhythms in E-box binding by CLK-CYC are dependent on the circadian clock. (A) ChIP assays were performed on wild-type flies collected at the indicated circadian times (CTs) during DD. PCR amplification products representing levels of the per CRS E-box bound are presented as described in Figure 1A, except that CLK-CYC binding was assayed exclusively by CLK immunoprecipitation. (B) ChIP assays were performed as described for A, except that relative levels of the tim UP E-box are shown. (C) ChIP assays were performed on _cyc_01 flies collected at the indicated times. PCR amplification products representing levels of the per CRS E-box bound are presented as described in A. (D) ChIP assays were performed as described for C, except that relative levels of the tim UP E-box are shown. (E) ChIP assays were performed on _per_01 flies collected at the indicated times during LD cycles. PCR amplification products representing levels of the per CRS E-box bound are presented as described in A. (F) ChIP assays were performed as described for E, except that relative levels of the tim UP E-box are shown. All ChIP experiments described above were independently repeated with similar results.

Figure 3.

The state of CLK phosphorylation cycles in a clock-dependent manner. (A) Western containing RBS extracts from wild-type and _Clk_Jrk fly heads. Wild-type flies were collected at the indicated times, and _Clk_Jrk was collected at ZT2. Hyperphosphorylated CLK (hyperP-CLK) and hypophosphorylated CLK (hypoP-CLK) run as broad bands at ∼150 kDa and 120 kDa, respectively. A nonspecific (ns) band, shown in a longer exposure below, indicates the relative amount of protein loaded in each lane. (B) RBS extracts from the heads of wild-type flies collected at the indicated times were either treated (+) or not treated (−) with phosphatase. In addition to the hyperP-CLK and hypoP-CLK bands, a nonphosphorylated CLK (nonP-CLK) band is shown. Extracts from _Clk_Jrk flies serve as a negative control for CLK. Two nonspecific (ns) bands are detected in these extracts. (C) Quantification of hypophosphorylated (dashed gray line), hyperphosphorylated (dashed black line), and total (solid black line) CLK abundance during an LD cycle. In each case, the peak level within a time course was normalized to 1.0. Results are based on three independent time courses. Error bars represent SEM. (D) RBS extracts from the heads of _per_01, _cyc_01, and wild-type flies collected at the indicated times were used to determine CLK levels and phosphorylation state.

Figure 4.

CLK abundance and phosphorylation cycle in ARK flies. (A) Western containing RBS extracts from the heads of ARK flies collected at the indicated times and _Clk_Jrk flies were probed with HA antibody. Hyperphosphorylated HACLK (hyperP-HACLK) and hypophosphorylated HACLK (hypoP-HACLK) bands are denoted. Note that this gel was not run or exposed as long as those in C and D, thus separation between CLK forms is not as great and weak bands are not as apparent. (B) Quantification of total HA-CLK levels in ARK flies. In each case, the peak level within a time course was normalized to 1.0. Results are based on three independent time courses. Error bars represent SEM. (C) RBS extracts from the heads of ARK flies collected at the indicated times were either treated (+) or not treated (−) with phosphatase. In addition to the hyperP-HACLK and hypoP-HACLK bands, a nonphosphorylated HACLK (nonP-HACLK) band is shown. Extracts from _Clk_Jrk flies serve as a negative control for HACLK. (D) RBS extracts from the heads of _per_01;ARK and ARK flies collected at the indicated times were used to determine HACLK levels and phosphorylation state. Extracts from _Clk_Jrk flies serve as a negative control for HACLK. Hyperphosphorylated HACLK (hyperP-HACLK) and hypophosphorylated HACLK (hypoP-HACLK) bands are denoted.

Figure 5.

DBT forms PER-dependent complexes with CLK. EB3-S extracts were prepared from the heads of wild-type (WT), _per_01, and _Clk_Jrk flies collected at the indicated times. Extracts were immunoprecipitated with DBT antisera (DBT) or rat serum (RS) or were used directly (input) to prepare Western blots. These Westerns were probed with CLK antiserum to detect hyperphosphorylated CLK (hyperP-CLK) and hypophosphorylated CLK (hypoP-CLK).

Figure 6.

DBT destabilizes CLK. (A) S2 cells were treated with luciferase RNAi (luc RNAi) or dbt RNAi and incubated for the number of hours indicated. Western blots were prepared using RBS extracts from these cells and probed with DBT antisera. (B) S2 cells treated with luc RNAi or dbt RNAi were transfected with the indicated amounts of pAct-HA_Clk_. Western blots prepared using RBS extracts from these cells and from the heads of ARK flies collected at ZT2 and ZT14 were probed with HA antiserum to detect hyperphosphorylated HACLK (hyperP-HACLK) and hypophosphorylated HACLK (hypoP-HACLK). (C) Untreated (−) and dbt RNAi-treated (+) S2 cells were transfected with the indicated amounts of pAct-HA_Clk_ and pAct-per. Western blots were prepared as described in B and probed with PER antiserum to reveal hyperphosphorylated PER (hyperP-PER) and hypophosphorylated PER (hypoP-PER). (D) RBS extracts were prepared from _dbt_ar/TM3,Sb (_dbt_ar/+), _dbt_P/TM3,Sb (_dbt_P/+), and _dbt_P/_dbt_ar flies collected at the indicated times. _dbt_ar/+ and _dbt_P/+ serve as controls for _dbt_P/_dbt_ar flies, and _Clk_Jrk serves as a negative control for CLK. The hyperphosphorylated CLK (hyperP-CLK) and hypophosphorylated CLK (hypoP-CLK) bands are denoted.

Figure 7.

Model for the regulation of circadian transcription in Drosophila. PER (blue hexagon) is generated during the early evening, but DBT (purple diamond) binding and phosphorylation (P) and possibly CK2 (green parallelogram) phosphorylation lead to PER degradation (stippled blue hexagon). TIM (yellow hexagon) binds to and stabilizes PER, and phosphorylation of PER by CK2 and TIM by SGG (pink rectangle) promotes nuclear localization of the PER-TIM-DBT complex. Nuclear PER-TIM-DBT complexes then bind to and remove hypophosphorylated CLK (red polygon)-CYC (green polygon) heterodimers from E-boxes (gray rectangle) to inhibit transcription. Coincident with transcriptional inhibition and PER-TIM-DBT-CLK-CYC complex formation, DBT phosphorylates PER and CLK to produce unstable hyperphosphorylated forms of PER and CLK. Degradation of hyperphosphorylated PER (stippled blue hexagon) and hyperphosphorylated CLK (stippled red polygon) and tyrosine phosphorylated TIM (stippled yellow hexagon) release DBT and CYC, and enable the accumulation of hypophosphorylated CLK to begin the next round of E-box binding and transcriptional activation. (Arrows) Subsequent steps in the process; (bar) inhibitory interaction; (wavy line) transcriptional activity; (double line) nuclear envelope.

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PER-dependent rhythms in CLK phosphorylation and E-box binding regulate circadian transcription - PubMed (original) (raw)