CK2 Inhibits TIMELESS Nuclear Export and Modulates CLOCK Transcriptional Activity to Regulate Circadian Rhythms

Cai, Yufeng; Xue, Yongbo; Truong, Cindy C.; Carmen-Li, Jose Del; Ochoa, Christopher; Vanselow, Jens T.; Murphy, Katherine A.; Li, Ying H.; Liu, Xianhui; Kunimoto, Ben L.; Zheng, Haiyan; Zhao, Caifeng; Zhang, Yong; Schlösser, Andreas; Chiu, Joanna C.

doi:10.1016/j.cub.2020.10.061

Cited by 16 publications

(16 citation statements)

References 94 publications

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“…This could be explained by the substitution to Aspartate not fully mimicking the structure of a phosphorylated Serine, or that constantly mimicking S47 phosphorylation impacts thermal compensation of the clock through a different mechanism than the S47A substitution. Notably it is not uncommon that phosphomimetic mutations do not produce opposite phenotype than phosphoinhibitory mutations, including in circadian proteins ( Lin et al, 2005 ; Chiu et al, 2011 ; Top et al, 2018 ; Cai et al, 2021 ). Thus, S47D could lead to overcompensation via yet unknown, different mechanism.…”

Section: Discussionmentioning

confidence: 99%

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PERIOD Phosphoclusters Control Temperature Compensation of the Drosophila Circadian Clock

Joshi

Cai²,

Xia³

et al. 2022

Front. Physiol.

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Ambient temperature varies constantly. However, the period of circadian pacemakers is remarkably stable over a wide-range of ecologically- and physiologically-relevant temperatures, even though the kinetics of most biochemical reactions accelerates as temperature rises. This thermal buffering phenomenon, called temperature compensation, is a critical feature of circadian rhythms, but how it is achieved remains elusive. Here, we uncovered the important role played by the Drosophila PERIOD (PER) phosphodegron in temperature compensation. This phosphorylation hotspot is crucial for PER proteasomal degradation and is the functional homolog of mammalian PER2 S478 phosphodegron, which also impacts temperature compensation. Using CRISPR-Cas9, we introduced a series of mutations that altered three Serines of the PER phosphodegron. While all three Serine to Alanine substitutions lengthened period at all temperatures tested, temperature compensation was differentially affected. S44A and S45A substitutions caused undercompensation, while S47A resulted in overcompensation. These results thus reveal unexpected functional heterogeneity of phosphodegron residues in thermal compensation. Furthermore, mutations impairing phosphorylation of the pers phosphocluster showed undercompensation, consistent with its inhibitory role on S47 phosphorylation. We observed that S47A substitution caused increased accumulation of hyper-phosphorylated PER at warmer temperatures. This finding was corroborated by cell culture assays in which S47A slowed down phosphorylation-dependent PER degradation at high temperatures, causing PER degradation to be excessively temperature-compensated. Thus, our results point to a novel role of the PER phosphodegron in temperature compensation through temperature-dependent modulation of the abundance of hyper-phosphorylated PER. Our work reveals interesting mechanistic convergences and differences between mammalian and Drosophila temperature compensation of the circadian clock.

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Section: Discussionmentioning

confidence: 99%

“…Protein extractions from Drosophila S2 cells and adult fly heads, western blotting, and image analysis was performed as previously described ( Chiu et al, 2008 ; Cai et al, 2021 ). 2–5 day old flies were entrained to 12:12 light-dark cycle for 3 days at indicated temperature.…”

Section: Discussionmentioning

confidence: 99%

PERIOD Phosphoclusters Control Temperature Compensation of the Drosophila Circadian Clock

Joshi

Cai²,

Xia³

et al. 2022

Front. Physiol.

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“…We show that CLK promotes BRM stability when both proteins are co-expressed in S2 cells, and this stability may be a result of CLK promoting the phosphorylation of BRM ( S1 Fig) . We speculate that CK2, a kinase that regulates PER-TIM nuclear accumulation [18,25,66–69] and phosphorylates CLK [29], may phosphorylate BRM to promote its stability given that CK2α phosphorylates BRG1 in mice [70,71]. Thus, phosphorylation of BRM by CLK-recruited CK2 could stabilize BRM protein levels, promoting its activity at clock loci.…”

Section: Discussionmentioning

confidence: 99%

“…Total RNA extraction was performed as described in [25], and cDNA synthesis and quantitative RT-PCR analysis was performed as described in [54]. The primer sets used to detect brm, cbp20, and per are listed in S1 Table . Each experiment consists of at least 3 biological replicates, and at least 2 technical replicates were performed for each biological replicate.…”

Section: Steady-state Mrna Analysismentioning

confidence: 99%

“…Around midnight, when PER and TIM levels accumulate to sufficient levels, they heterodimerize and translocate into the nucleus [16][17][18], where they interact with the CLK-CYC complex to repress their own transcription and the transcription of other CLK-activated genes [8,19,20]. Finally, proteasome dependent degradation of PER and TIM [13,[21][22][23] and modulation of CLK activity by post-translational modifications [24][25][26][27][28][29] terminates the circadian repression phase in late day to early morning, initiating the next circadian cycle.…”

Section: Introductionmentioning

confidence: 99%

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CLOCK and TIMELESS regulate rhythmic occupancy of the BRAHMA chromatin-remodeling protein at clock gene promoters

Tabuloc

Kwok

Chan

et al. 2022

Preprint

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Circadian clock and chromatin remodeling complexes are tightly intertwined systems that regulate rhythmic gene expression. The circadian clock promotes rhythmic expression, timely recruitment, and/or activation of chromatin remodelers, while chromatin remodelers regulate accessibility of clock transcription factors to the DNA to influence expression of clock genes. We previously reported that the BRAHMA (BRM) chromatin remodeling complex promotes the repression of circadian gene expression inDrosophila.In this study, we investigated the mechanisms by which the circadian clock feeds back to modulate daily BRM activity. Using chromatin immunoprecipitation, we observed rhythmic BRM binding to clock gene promoters despite constitutive BRM protein expression, suggesting that factors other than protein abundance are responsible for rhythmic BRM occupancy at clock-controlled loci. Since we previously reported that BRM interacts with two key clock proteins, CLOCK (CLK) and TIMELESS (TIM), we examined their effect on BRM occupancy to theperiod (per)promoter. We observed reduced BRM binding to the DNA inclknull flies, suggesting that CLK is involved in enhancing BRM occupancy to initiate transcriptional repression at the conclusion of the activation phase. Additionally, we observed reduced BRM binding to theperpromoter in flies overexpressing TIM, suggesting that TIM promotes BRM removal from DNA. This conclusion is further supported by elevated BRM binding to theperpromoter in flies subjected to constant light. In summary, this study provides new insights into the reciprocal regulation between the circadian clock and the BRM chromatin remodeling complex.Author SummaryCircadian clocks are endogenous time-keeping mechanisms that allow organisms to anticipate and adapt to daily changes in their external environment. These clocks are driven by a molecular oscillator that generates rhythms in the expression of many genes, termed clock-controlled genes. The genomic DNA containing these clock-controlled genes are also modified in a rhythmic manner throughout the day. DNA are more tightly packaged with histone proteins when transcription of clock-controlled genes is repressed while the interaction between DNA and histone proteins are more relaxed during transcriptional activation. We found that two key clock proteins, CLOCK and TIMELESS, regulate daily rhythmicity in the binding of BRAHMA, a chromatin remodeler, to DNA spanning clock-controlled genes to facilitate their rhythmic gene expression cycles. Moreover, because TIMELESS is sensitive to light, our study provides new insights into how light can affect DNA structure and gene expression.

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Assaying Circadian Locomotor Activity Rhythm in Drosophila

et al. 2022

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CK2 Inhibits TIMELESS Nuclear Export and Modulates CLOCK Transcriptional Activity to Regulate Circadian Rhythms

Cited by 16 publications

References 94 publications

PERIOD Phosphoclusters Control Temperature Compensation of the Drosophila Circadian Clock

PERIOD Phosphoclusters Control Temperature Compensation of the Drosophila Circadian Clock

CLOCK and TIMELESS regulate rhythmic occupancy of the BRAHMA chromatin-remodeling protein at clock gene promoters

Assaying Circadian Locomotor Activity Rhythm in Drosophila

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