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Circadian Repressors CRY1 and CRY2 Broadly Interact with Nuclear Receptors and Modulate Transcriptional Activity

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Circadian Repressors CRY1 and CRY2 Broadly Interact with Nuclear Receptors and Modulate Transcriptional Activity Circadian repressors CRY1 and CRY2 broadly interact with nuclear receptors and modulate transcriptional activity Anna Kriebsa, Sabine D. Jordana, Erin Sotoa, Emma Henrikssona,b, Colby R. Sandatea, Megan E. Vaughana, Alanna B. Chana, Drew Duglana, Stephanie J. Pappa, Anne-Laure Hubera, Megan E. Afetiana, Ruth T. Yuc, Xuan Zhaoc, Michael Downesc, Ronald M. Evansc,d,e, and Katja A. Lamiaa,e,1 aDepartment of Molecular Medicine, The Scripps Research Institute, La Jolla, CA 92037; bDepartment of Clinical Sciences, Clinical Research Centre, Lund University, Malmö 20502, Sweden; cGene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037; dHoward Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, CA 92037; and eCenter for Circadian Biology, University of California, San Diego, CA 92161 Edited by Steven A. Kliewer, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX, and approved July 3, 2017 (received for review March 24, 2017) Nuclear hormone receptors (NRs) regulate physiology by sensing NRs are intimately connected to the circadian clock and its lipophilic ligands and adapting cellular transcription appropriately. function in adapting daily metabolic outputs to the 24-h day/ A growing understanding of the impact of circadian clocks on night cycle. The NRs REV-ERBα/β and RORα/γ are key regu- mammalian transcription has sparked interest in the interregula- lators of core clock function (4), and many NRs are rhythmically tion of transcriptional programs. Mammalian clocks are based on a expressed (5). PER, CRY, and CLOCK can regulate NRs by di- – transcriptional feedback loop featuring the transcriptional activators verse mechanisms (6 9). These and indirect effects, like rhythmic circadian locomotor output cycles kaput (CLOCK) and brain and abundance of endogenous ligands and the rhythmic transcription muscle ARNT-like 1 (BMAL1), and transcriptional repressors cryp- of coactivators and corepressors, convey time-of-day information tochrome (CRY) and period (PER). CRY1 and CRY2 bind independently to NR-regulated pathways, such as lipid, glucose, and xenobiotic metabolism (4). of other core clock factors to many genomic sites, which are enriched Upon binding of a xenobiotic ligand, the NRs pregnane X re- for NR recognition motifs. Here we report that CRY1/2 serve as co- ceptor (PXR) and constitutive androstane receptor (CAR) induce repressors for many NRs, indicating a new facet of circadian control expression of proteins required for xenobiotic detoxification. Xe- of NR-mediated regulation of metabolism and physiology, and spe- nobiotic metabolism is subject to time-of-day–dependent regula- cifically contribute to diurnal modulation of drug metabolism. tion: in humans, the half-life of CYP3A substrates is shortest in the afternoon (10); in rodents, the lethal toxicity of a fixed dose of nuclear hormone receptor | cryptochrome | circadian | corepressor | a drug depends on the time of administration (11). The circadian xenobiotic metabolism transcriptome (6, 7) and proteome (8) are enriched for compo- nents of xenobiotic detoxification pathways. Here we report a – uclear receptors (NRs) regulate transcription in response to comprehensive survey of CRY NR interactions and enhanced Nlipophilic ligands (1). Most NRs contain four domains: The metabolism of the anesthetic ketamine in CRY-deficient mice. N-terminal activation function 1 (AF1) is followed by a highly Results conserved DNA binding domain (DBD), which is connected to CRY1 Interacts with Many NRs. the ligand binding domain (LBD) via a flexible hinge region. The We examined the interaction of all mouse NRs with mouse CRY1 using coimmunoprecipitation LBD contains the hydrophobic ligand binding pocket and activa- (co-IP) (Fig. 1A). Approximately one-third of mouse NRs con- tion function 2 (AF2) located in helix 12 (H12). Structural rear- sistently interact with CRY1, whereas another third is weakly or rangement of H12 is crucial for ligand-dependent transactivation. variably associated with CRY1 (Fig. 1B). The strongest interactors Regulation of transcription by NRs depends on interaction with include steroid hormone receptors, lipid-sensing peroxisome corepressors, such as nuclear receptor corepressor (NCoR) or silencing mediator for retinoid or thyroid-hormone receptors Significance (SMRT), and coactivators, such as peroxisome proliferator-activated receptor-γ coactivator 1⍺ (PGC1⍺) or steroid receptor coactivators. Nuclear receptors (NRs) are ligand-sensing transcription factors Steroid hormone receptors are enriched in the cytoplasm in the that are crucial for the proper regulation of mammalian de- absence of ligand. Upon agonist ligand binding, they translocate velopment, physiology, and metabolism. Their ligand-binding to the nucleus and bind DNA. Recruitment of coactivators then capability makes NRs attractive drug targets, but can also lead allows transcription to occur. Conversely, nonsteroid-binding NRs to the adverse side effects of prescription drugs. Our research are bound to DNA and corepressors in the absence of ligand. contributes to a better understanding of how NRs are regu- Binding of agonist ligand displaces corepressors in favor of coac- lated in a time-of-day–dependent manner by a component of tivators, allowing transcription initiation. the circadian clock, cryptochrome, and is foundational to fur- Mammalian circadian clocks synchronize physiology and me- ther research aiming to make drug administration routines tabolism with daily environmental changes. The mammalian clock more effective and safer. is based on a transcription–translation feedback loop featuring the transcription factors circadian locomotor output cycles kaput Author contributions: A.K., R.M.E., and K.A.L. designed research; A.K., S.D.J., E.S., E.H., (CLOCK), brain and muscle ARNT-like 1 (BMAL1), crypto- C.R.S., M.E.V., A.B.C., D.D., S.J.P., A.-L.H., M.E.A., and K.A.L. performed research; A.K., X.Z., chrome (CRY), and period (PER) (2). Forming a heterodimer, and M.D. contributed new reagents/analytic tools; A.K., R.T.Y., and K.A.L. analyzed data; CLOCK and BMAL1 drive transcription of target genes, in- and A.K. and K.A.L. wrote the paper. cluding those encoding their own repressors period (PER1, PER2, The authors declare no conflict of interest. PER3) and cryptochrome (CRY1, CRY2). PER and CRY di- This article is a PNAS Direct Submission. merize and repress CLOCK and BMAL1, allowing the cycle to 1To whom correspondence should be addressed. Email: [email protected]. repeat. This core clock directly or indirectly drives oscillating This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. transcription of ≈43% of all protein coding genes (3). 1073/pnas.1704955114/-/DCSupplemental. 8776–8781 | PNAS | August 15, 2017 | vol. 114 | no. 33 www.pnas.org/cgi/doi/10.1073/pnas.1704955114 Downloaded by guest on September 25, 2021 Fig. 1. CRY1 interacts with a subset of nuclear re- ceptors. (A) Co-IP of FLAG-CRY1 with V5-NRs tran- siently expressed in HEK293T cells. (B) Table listing strongly or weakly interacting NRs, and NRs that were notoronlypoorlyexpressed. proliferator-activated receptors (PPARs), vitamin D receptor clock. Our results and those of others (9, 15) suggest that CRYs (VDR), and the xenobiotic receptors PXR and CAR. may function independently of other core clock proteins to CRY1 and CRY2 bind many genomic sites independent of regulate NR-driven transcription. NR corepressors are recruited other clock proteins, and these unique sites were enriched for to the LBD, through a conserved hydrophobic motif, the co- NR-binding motifs (9). Using these previously reported datasets repressor NR box (CoRNR box), comprising I/L-X-X-I/V-I to perform motif analysis for CRY1 and CRY2 genomic binding sequences (18, 19). Corepressors dissociate following a confor- BIOCHEMISTRY sites, we detected consensus sites of liver-expressed NRs, including mational change of H12 caused by agonist ligand binding (20). hepatocyte nuclear factor 4-α (HNF4α), PPAR, Rev-ERB, far- PXR, because of its crucial role in drug metabolism, has been nesoid X receptor (FXR), and retinoic acid receptor (RAR) extensively studied structurally. Potent and specific synthetic (Datasets S1 and S2). Comparing CRY1 and CRY2 genomic agonist ligands are available, making PXR a prime candidate to binding sites with those published for glucocorticoid receptor explore the biochemical features of the interaction with CRY. (GR) (12), Rev-ERBα,Rev-ERBβ (13), and PPARα (14) (Fig. 2A), To determine whether the LBD of PXR is sufficient for the we detected overlap of CRY1 and CRY2 binding with up to 37% of interaction with CRYs, we performed co-IP of full-length CRY1 NR binding sites (Fig. 2B). These sites include Pck1 (GR) (Datasets and CRY2 with full-length (FL) PXR or the PXR LBD, and S3 and S4), confirming previous reports (15), Pdk4 (PPARα)(Dataset observed that the LBD is sufficient for interaction (Fig. 4A). S5), consistent with our finding that CRYs regulate PPARδ Furthermore, recombinant CRY2 (amino acids 1–512) interacts and Pdk4 in muscle (16), and Bmal1 (Rev-ERBα,Rev-ERBβ) directly with recombinant PXR LBD (Fig. 4B). However, the (Datasets S6 and S7), suggesting that CRYs could contribute to affinity of the interaction is weak, possibly as a result of Bmal1 transcriptional regulation. CRY2 lacking the C-terminal tail, which we and others have thus Many NRs display increased affinity for CRY2 compared with far been unable to express and purify (21, 22) (Fig. 4C). CRYs CRY1, allowing us to use CRY1/2 hybrid constructs (17) (Fig. contain an amino acid sequence that resembles a CoRNR box, in the 3A) to identify the domains required for preferential interaction. PHR domain. However, mutating this motif does not disrupt in- Co-IP of CRY hybrids with PXR and CAR revealed that the A teraction with PXR as similar mutations in NCOR and SMRT do (18, and B domains, which correspond to the photolyase homology 19, 23), suggesting that it is not required for interaction of CRY with region (PHR), as well as the D domain, which mostly consists of NRs (Fig.
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