Nuclear receptor HNF4A transrepresses CLOCK: BMAL1 and modulates tissue-specific circadian networks Meng Qua, Tomas Duffyb, Tsuyoshi Hirotac, and Steve A. Kaya,1 aKeck School of Medicine, University of Southern California, Los Angeles, CA 90089; bDepartment of Molecular Medicine, The Scripps Research Institute, La Jolla, CA 92037; and cInstitute of Transformative Bio-Molecules, Nagoya University, 464-8602 Nagoya, Japan Contributed by Steve A. Kay, November 6, 2018 (sent for review September 24, 2018; reviewed by Carla B. Green and John B. Hogenesch) Either expression level or transcriptional activity of various nuclear NRs canonically function as ligand-activated transcription receptors (NRs) have been demonstrated to be under circadian factors that regulate the expression of their target genes to control. With a few exceptions, little is known about the roles of affect physiological pathways (19). The importance of NRs in NRs as direct regulators of the circadian circuitry. Here we show maintaining optimal physiological homeostasis is illustrated in that the nuclear receptor HNF4A strongly transrepresses the their identification as potential targets for therapeutic drug transcriptional activity of the CLOCK:BMAL1 heterodimer. We development to combat a diverse array of diseases, including define a central role for HNF4A in maintaining cell-autonomous reproductive disorders, inflammation, cancer, diabetes, car- circadian oscillations in a tissue-specific manner in liver and colon diovascular disease, and obesity (20). Various NRs have been cells. Not only transcript level but also genome-wide chromosome implicated as targets of the circadian clock, which may con- binding of HNF4A is rhythmically regulated in the mouse liver. tribute to the circadian regulation of nutrient and energy me- ChIP-seq analyses revealed cooccupancy of HNF4A and CLOCK: tabolism. Over half of the NR family members are expressed in BMAL1 at a wide array of metabolic genes involved in lipid, a rhythmic manner (21). Further, PER2 and CRYs were found glucose, and amino acid homeostasis. Taken together, we estab- to broadly interact with NRs and potentially modulate their lish that HNF4A defines a feedback loop in tissue-specific mamma- – lian oscillators and demonstrate its recruitment in the circadian transcriptional activity (22 25). regulation of metabolic pathways. Apart from the circadian control of NR expression and tran- scriptional activity, other direct links between NR activity and ’ circadian rhythm | nuclear receptor | HNF4A | CLOCK:BMAL1 | circadian clock function have been identified via NRs role as transrepression circadian clock inputs. In addition to functions in development, metabolism, and immunity, the nuclear receptors REV-ERBs and retinoic acid receptor-related orphan receptors (RORs) ircadian rhythms are intrinsic, entrainable oscillations of constitute the secondary feedback loop in the circadian cycle, by Cabout 24 h in behavior, metabolism, and physiology found in Bmal1 almost all living organisms. This internal clock allows organisms directly repressing and activating expression, respectively, to synchronize their physiology and behavior with the predictable and in turn being transcriptionally regulated by CLOCK:BMAL1 cycle of day and night. Disruption of normal circadian rhythms leads to clinically relevant disorders, including neurodegeneration, Significance diabetes, obesity, and cardiovascular disease (1–4). In mammals, the core circadian circuit composed of transcriptional activators Interlocked feedback loops promote robustness and stability in and repressors that form an autoregulatory transcriptional feed- a system and are a feature of circadian clocks in both animal back loop is necessary for the generation and regulation of cir- and plants. The mammalian circadian clock is known to consist cadian rhythms with an endogenous period close to 24 h. The of two transcriptional feedback loops, relying on the tran- bHLH-PAS transcription factors (TFs) CLOCK and BMAL1 are scriptional activity of the master complex CLOCK:BMAL1 and heterodimeric transcriptional activators that drive the expression the feedback regulation by its target genes. Our research ex- of Period genes (Per1, Per2,andPer3)andCryptochrome genes tends this knowledge by proposing a feedback loop in pe- (Cry1 and Cry2), by binding E-box cis-regulatory elements at ripheral circadian oscillators and highlights the underlying promoters (5–9). Subsequently, the PER–CRY protein complex mechanisms mediated by the unappreciated CLOCK:BMAL1 inhibits transcription of its own genes by directly inhibiting transrepression activity of the circadian nuclear receptor HNF4A. GENETICS CLOCK:BMAL1 activity (8, 10–13). This feedback loop has Further, our data suggest that the hepatic roles of HNF4A been known as the critical hub of the mammalian oscillator. are circadian. While many mammalian peripheral tissues have circadian clocks, genes showing circadian expression are markedly diverse Author contributions: M.Q. and S.A.K. designed research; M.Q. performed research; M.Q., within individual tissues of mouse or human, indicating tissue- T.D., and T.H. analyzed data; and M.Q., T.H., and S.A.K. wrote the paper. specific regulation of circadian output relevant to the function of Reviewers: C.B.G., The University of Texas Southwestern Medical Center; and J.B.H., Cincinnati Children’s Hospital Medical Center. the particular organ (14–18). The core clock may be regulated in Conflict of interest statement: S.A.K., J.B.H., and C.B.G. are coauthors on a 2017 guideline a tissue-specific manner as well, supported by observations that article. explants of diverse mouse tissues express differences in circadian This open access article is distributed under Creative Commons Attribution-NonCommercial- period and phase (16), and that output networks must be driven NoDerivatives License 4.0 (CC BY-NC-ND). by the core clock components in a cell type-specific manner. Data deposition: HNF4A ChIP-seq data at ZT4 and ZT16 have been deposited in the Gene However, such tissue-specific factors responsible for robust Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. tissue-specific circadian rhythmicity remain to be identified. GSE118007). The nuclear receptor (NR) superfamily is composed of 48 mem- 1To whom correspondence should be addressed. Email: [email protected]. bers in human. NRs coordinate processes as diverse as devel- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. opment, reproduction, and many aspects of metabolism. As 1073/pnas.1816411115/-/DCSupplemental. sensors for lipophilic vitamins, hormones, and dietary lipids, Published online December 10, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1816411115 PNAS | vol. 115 | no. 52 | E12305–E12312 Downloaded by guest on October 2, 2021 (26–29). Dysregulation of REV-ERBs and RORs have dramatic core clock proteins and the HNF4A protein, as shown in coimmu- effects on the robustness of circadian oscillators (30–34). How- noprecipitation experiments using either recombinant or endoge- ever, little is known as to whether other NRs are involved in the nous proteins (Fig. 1 A–C). To evaluate the biological significance of regulation of core clock parameters. the interactions, we hypothesized that either the core clock The nuclear receptor hepatocyte nuclear factor 4A (HNF4A) components regulate the activity of nuclear receptor HNF4A, or binds as a homodimer to the consensus DNA sequence consisting HNF4A may transduce metabolic signals as inputs to the circadian of two direct repeats and controls the expression of downstream system. To test the first hypothesis, we conducted a reporter assay genes (35). It is expressed in a tissue-specific manner and plays in HEK 293T cells using a luciferase reporter driven by tandem essential roles in the development and physiology of liver, pan- HNF4A consensus binding motifs. Neither CRY1 nor CLOCK: creas, kidney, and intestines, particularly lipid and glucose me- BMAL1 coexpression affected the HNF4A transcriptional activ- tabolism and inflammation (36–42). In the current study, we ity, indicating that HNF4A activity is unlikely to be directly reg- demonstrate that HNF4A potently inhibits the transcriptional ulated by these clock proteins (SI Appendix,Fig.S1). activity of CLOCK:BMAL1 heterodimer. Furthermore, our data We then tested the second hypothesis, asking whether the core clearly show that HNF4A is necessary for the regulation of in- clock feedback loops are regulated by the HNF4A protein. As trinsic circadian oscillations in liver and colon cells, suggesting its HNF4A consistently binds CLOCK:BMAL1, we evaluated the role in regulating tissue-specific clock networks. effect of HNF4A on CLOCK:BMAL1 activity in endpoint reporter assays using the E-box-dLuc, Per1-Luc,orPer2-dLuc luciferase re- Results porters. Surprisingly, we observed a consistent inhibition of CLOCK: Identification of HNF4A as a CLOCK:BMAL1 Transrepressor. The BMAL1 activity by HNF4A coexpression in a dose-dependent pat- growing body of evidence demonstrating a link between nuclear tern (P < 0.05), similar to the canonical clock repressor CRY1 (Fig. 1 receptors, metabolic pathways, and intrinsic circadian rhyth- D–F). Collectively, these findings indicate activity of the nuclear re- micity (21–25) prompted us to test the potential for a broader ceptor HNF4A in transrepressing another transcription factor range of nuclear receptors in mediating this cross-talk. In a CLOCK:BMAL1