A positive feedback loop links circadian clock factor CLOCK-BMAL1 to the basic transcriptional machinery

Abstract

Circadian clocks in mammals are built on a negative feedback loop in which the heterodimeric transcription factor circadian locomotor output cycles kaput (CLOCK)-brain, muscle Arnt-like 1 (BMAL1) drives the expression of its own inhibitors, the PERIOD and CRYPTOCHROME proteins. Reactivation of CLOCK-BMAL1 occurs at a specific time several hours after PERIOD and CRYPTOCHROME protein turnover, but the mechanism underlying this process is unknown. We found that mouse BMAL1 complexes include TRAP150 (thyroid hormone receptor-associated protein-150; also known as THRAP3). TRAP150 is a selective coactivator for CLOCK-BMAL1, which oscillates under CLOCK-BMAL1 transcriptional control. TRAP150 promotes CLOCK-BMAL1 binding to target genes and links CLOCK-BMAL1 to the transcriptional machinery at target-gene promoters. Depletion of TRAP150 caused low-amplitude, long-period rhythms, identifying it as a positive clock element. The activity of TRAP150 defines a positive feedback loop within the clock and provides a potential mechanism for timing the reactivation of circadian transcription. C ircadian clocks are endogenous oscillators that drive daily rhythms of physiology and behavior. The mammalian clock, intrinsic to most cells and tissues (1, 2), is built on a conserved negative feedback loop that generates circadian rhythms at the molecular level (3). The core positive element of the clock is the heterodimeric transcription factor circadian locomotor output cycles kaput (CLOCK)-brain, muscle Arnt-like 1 (BMAL1), which drives transcription of Period (Per) and Cryptochrome (Cry) genes from E-box sites (4). PER and CRY proteins, acting as negative elements of the clock, enter the nucleus, associate with CLOCK-BMAL1 (5) at E-box sites (6), and suppress the transcriptional activity of CLOCK-BMAL1 in part by recruiting the SIN3-HDAC histone deacetylase complex (6) and inhibiting transcriptional termination (7). Turnover of PERs and CRYs ends the negative-feedback phase of the cycle (8-11). An interlocked feedback loop involving REV-ERBα and -β (nuclear receptor subfamily 1, group D, members 1 and 2, respectively) contributes to clock function (12)(13)(14).Reactivation of CLOCK-BMAL1 transcription of circadian target genes occurs several hours after the end of negative feedback (15,16), suggesting that the onset of circadian transcription in each cycle by CLOCK-BMAL1 is not simply a passive consequence of the turnover of negative-feedback proteins, but is positively regulated and timed by unknown clock-controlled factors. Evidence that there is active positive regulation of CLOCK-BMAL1 comes from reports showing enhancement of CLOCK-BMAL1 transcriptional activity by chromatin-modifying proteins by CBP/p300 (17), MLL1 (18), and JARID1a (19). Although not previously described, a clock-controlled, rhythmic positive factor for CLOCK-BMAL1 would provide a potential mechanism for precisely setting the onset of transcription each circadian cycle. ResultsTo identify factors associated with CLOCK-BMAL1, we used FLAG antibodies to affinit...

Vertebrate-like CRYPTOCHROME 2 from monarch regulates circadian transcription via independent repression of CLOCK and BMAL1 activity

“…Alternatively, given that our results were obtained in a cellular context rather than in vitro chemical shift perturbation studies (14), it is possible that additional coactivators bound to dpCLK: dpBMAL1 also compete with dpCRY2 for binding. Histonemodifying enzyme orthologs of MLL and JARID1a, which are recruited at CLOCK:BMAL1, or proteins that recruit the transcriptional machinery are all potential candidates (34)(35)(36)(37).…”

Thrap3 docks on phosphoserine 273 of PPARγ and controls diabetic gene programming

“…Another study showed that Thrap3 could interact with PPARg, and Thrap3 expression was required for adipose differentiation (Katano-Toki et al 2013). More recent studies have demonstrated that Thrap3 is a selective transcriptional coactivator for CLOCK-BMAL1, a common circadian clock factor, and depletion of Thrap3 causes low-amplitude, long-period circadian rhythms, identifying it as a positive clock element (Lande-Diner et al 2013). In addition, it has been reported that phosphorylation of PSF/SFPQ by glycogen synthase kinase 3 (GSK3) promotes an interaction with Thrap3 and prevents PSF from binding to the CD45 promoter (Heyd and Lynch 2010).…”

“…Thrap3 specifically interacted with wild-type PPARg following TNF-a treatment but was not observed in association with PPARg S273A in cells treated with TNF-a. Thrap3 is known to be a coregulatory protein that is loosely associated with the Mediator complex (Fondell et al 1996;Ito et al 2002) and is also reported to regulate several transcription factors (Lande-Diner et al 2013). Thrap3 is therefore a plausible candidate for involvement in phosphorylation-mediated PPARg regulation.…”

Mistimed sleep disrupts circadian regulation of the human transcriptome

“…7D). THRAP3 has recently been identified as a coactivator of the CLOCK-BMAL1 complex and promotes its binding to target genes, linking it with the transcriptional machinery (38). This category also included the methyltransferase transcript MLL3, and the expression profiles of several transcripts associated with methylation are affected by mistimed sleep (Fig.…”