Circadian Amplitude Regulation via FBXW7-Targeted REV-ERBα Degradation.

Defects in circadian rhythm influence physiology and behavior with implications for the treatment of sleep disorders, metabolic disease, and cancer. Although core regulatory components of clock rhythmicity have been defined, insight into the mechanisms underpinning amplitude is limited. Here, we show that REV-ERBα, a core inhibitory component of clock transcription, is targeted for ubiquitination and subsequent degradation by the F-box protein FBXW7. By relieving REV-ERBα-dependent repression, FBXW7 provides an unrecognized mechanism for enhancing the amplitude of clock gene transcription. Cyclin-dependent kinase 1 (CDK1)-mediated phosphorylation of REV-ERBα is necessary for FBXW7 recognition. Moreover, targeted hepatic disruption of FBXW7 alters circadian expression of core clock genes and perturbs whole-body lipid and glucose levels. This CDK1-FBXW7 pathway controlling REV-ERBα repression defines an unexpected molecular mechanism for re-engaging the positive transcriptional arm of the clock, as well as a potential route to manipulate clock amplitude via small molecule CDK1 inhibition.

The interplay between the circadian clock and abiotic stress responses mediated by ABF3 and CCA1/LHY.

Climate change is a global concern for all life on our planet, including humans and plants. Plants' growth and development are significantly affected by abiotic stresses, including adverse temperature, inadequate or excess water availability, nutrient deficiency, and salinity. The circadian clock is a master regulator of numerous developmental and metabolic processes in plants. In an effort to identify new clock-related genes and outputs through bioinformatic analysis, we have revealed that CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) play a crucial role in regulating a wide range of abiotic stress responses and target ABSCISIC ACID RESPONSIVE ELEMENTS-BINDING FACTOR3 (ABF3), a key transcription factor in the plant hormone Abscisic acid (ABA)-signaling pathway. Specifically, we found that CCA1 and LHY regulate the expression of ABF3 under diel conditions, as well as seed germination under salinity. Conversely, ABF3 controls the expression of core clock genes and orchestrates the circadian period in a stress-responsive manner. ABF3 delivers the stress signal to the central oscillator by binding to the promoter of CCA1 and LHY. Overall, our study uncovers the reciprocal regulation between ABF3 and CCA1/LHY and molecular mechanisms underlying the interaction between the circadian clock and abiotic stress. This finding may aid in developing molecular and genetic solutions for plants to survive and thrive in the face of climate change.

GIGANTEA adjusts the response to shade at dusk by directly impinging on PHYTOCHROME INTERACTING FACTOR 7 function.

For plants adapted to bright light, a decrease in the amount of light received can be detrimental to their growth and survival. Consequently, in response to shade from surrounding vegetation, they initiate a suite of molecular and morphological changes known as the shade avoidance response through which stems and petioles elongate in search for light. Under sunlight-night cycles, the plant's responsiveness to shade varies across the day, being maximal at dusk time. While a role for the circadian clock in this regulation has long been proposed, mechanistic understanding of how it is achieved is incomplete. Here, we show that the clock component GIGANTEA (GI) directly interacts with the transcriptional regulator PHYTOCHROME INTERACTING FACTOR 7 (PIF7), a key player in the response to shade. GI represses PIF7 transcriptional activity and the expression of its target genes in response to shade, thereby fine-tuning the magnitude of the response to limiting light conditions. We find that under light/dark cycles, this function of GI is required to adequately modulate the gating of the response to shade at dusk. Importantly, we also show that this circuit primarily operates in epidermal cells, highlighting the relevance of tissue-specific clock-output connections for the regulation of plant development in resonance with the environment.

Circadian regulator BMAL1::CLOCK promotes cell proliferation in hepatocellular carcinoma by controlling apoptosis and cell cycle.

Hepatocellular carcinoma (HCC) remains a global health challenge whose incidence is growing worldwide. Previous evidence strongly supported the notion that the circadian clock controls physiological homeostasis of the liver and plays a key role in hepatocarcinogenesis. Despite the progress, cellular and molecular mechanisms underpinning this HCC-clock crosstalk remain unknown. Addressing this knowledge gap, we show here that although the human HCC cells Hep3B, HepG2, and Huh7 displayed variations in circadian rhythm profiles, all cells relied on the master circadian clock transcription factors, BMAL1 and CLOCK, for sustained cell growth. Down-regulating Bmal1 or Clock in the HCC cells induced apoptosis and arrested cell cycle at the G2/M phase. Mechanistically, we found that inhibiting Bmal1/Clock induced dysregulation of the cell cycle regulators Wee1 and p21 which cooperatively contribute to tumor cell death. Bmal1/Clock knockdown caused downregulation of Wee1 that led to apoptosis activation and upregulation of p21 which arrested the cell cycle at the G2/M phase. Collectively, our results suggest that the circadian clock regulators BMAL1 and CLOCK promote HCC cell proliferation by controlling Wee1 and p21 levels, thereby preventing apoptosis and cell cycle arrest. Our findings shed light on cellular impact of the clock proteins for maintaining HCC oncogenesis and provide proof-of-principle for developing cancer therapy based on modulation of the circadian clock.

CRY2 isoform selectivity of a circadian clock modulator with antiglioblastoma efficacy.

The mammalian cryptochrome isoforms, CRY1 and CRY2, are core circadian clock regulators that work redundantly. Recent studies revealed distinct roles of these closely related homologs in clock output pathways. Isoform-selective control of CRY1 and CRY2 is critical for further understanding their redundant and distinct roles. KL001 was the first identified small-molecule CRY modulator that activates both CRY1 and CRY2. SHP656 is an orally available KL001 derivative and has shown efficacy in blood glucose control and inhibition of glioblastoma stem cell (GSC) growth in animal models. However, CRY isoform selectivity of SHP656 was uncharacterized, limiting understanding of the roles of CRY1 and CRY2. Here, we report the elucidation of CRY2 selectivity of SHP656. SHP656 lengthened cellular circadian period in a CRY2-dependent manner and selectively interacted with CRY2. By determining the X-ray crystal structure of CRY2 in complex with SHP656 and performing molecular dynamics simulations, we elucidated compound interaction mechanisms. SHP656 binding was compatible with the intrinsic CRY2 gatekeeper W417 "in" orientation and also a close "further in" conformation. Perturbation of W417 interaction with the lid loop resulted in a reduced effect of SHP656 on CRY2, supporting an important role of gatekeeper orientation in isoform selectivity. We also identified the R form of SHP656 (called SHP1703) as the active isomer. Treatment with SHP1703 effectively reduced GSC viability. Our results suggest a direct role of CRY2 in glioblastoma antitumorigenesis and provide a rationale for the selective modulation of CRY isoforms in the therapeutic treatment of glioblastoma and other circadian clock-related diseases.

The actin cytoskeleton-MRTF/SRF cascade transduces cellular physical niche cues to entrain the circadian clock.

The circadian clock is entrained to daily environmental cues. Integrin-linked signaling via actin cytoskeleton dynamics transduces physical niche cues from the extracellular matrix to myocardin-related transcription factor (MRTF)/serum response factor (SRF)-mediated transcription. The actin cytoskeleton organization and SRF-MRTF activity display diurnal oscillations. By interrogating disparate upstream events in the actin cytoskeleton-MRTF-A/SRF signaling cascade, we show that this pathway transduces extracellular niche cues to modulate circadian clock function. Pharmacological inhibition of MRTF-A/SRF by disrupting actin polymerization or blocking the ROCK kinase induced period lengthening with augmented clock amplitude, and genetic loss of function of Srf or Mrtfa mimicked the effects of treatment with actin-depolymerizing agents. In contrast, actin polymerization shortened circadian clock period and attenuated clock amplitude. Moreover, interfering with the cell-matrix interaction through blockade of integrin, inhibition of focal adhesion kinase (FAK, encoded by Ptk2) or attenuating matrix rigidity reduced the period length while enhancing amplitude. Mechanistically, we identified that the core clock repressors Per2, Nr1d1 and Nfil3 are direct transcriptional targets of MRTF-A/SRF in mediating actin dynamics-induced clock response. Collectively, our findings defined an integrin-actin cytoskeleton-MRTF/SRF pathway in linking clock entrainment with extracellular cues that might facilitate cellular adaptation to the physical niche environment.

A genome-wide RNAi screen for modifiers of the circadian clock in human cells.

Two decades of research identified more than a dozen clock genes and defined a biochemical feedback mechanism of circadian oscillator function. To identify additional clock genes and modifiers, we conducted a genome-wide small interfering RNA screen in a human cellular clock model. Knockdown of nearly 1000 genes reduced rhythm amplitude. Potent effects on period length or increased amplitude were less frequent; we found hundreds of these and confirmed them in secondary screens. Characterization of a subset of these genes demonstrated a dosage-dependent effect on oscillator function. Protein interaction network analysis showed that dozens of gene products directly or indirectly associate with known clock components. Pathway analysis revealed these genes are overrepresented for components of insulin and hedgehog signaling, the cell cycle, and the folate metabolism. Coupled with data showing many of these pathways are clock regulated, we conclude the clock is interconnected with many aspects of cellular function.

A genome-wide microRNA screen identifies the microRNA-183/96/182 cluster as a modulator of circadian rhythms.

The regulatory mechanisms of circadian rhythms have been studied primarily at the level of the transcription-translation feedback loops of protein-coding genes. Regulatory modules involving noncoding RNAs are less thoroughly understood. In particular, emerging evidence has revealed the important role of microRNAs (miRNAs) in maintaining the robustness of the circadian system. To identify miRNAs that have the potential to modulate circadian rhythms, we conducted a genome-wide miRNA screen using U2OS luciferase reporter cells. Among 989 miRNAs in the library, 120 changed the period length in a dose-dependent manner. We further validated the circadian regulatory function of an miRNA cluster, miR-183/96/182, both in vitro and in vivo. We found that all three members of this miRNA cluster can modulate circadian rhythms. Particularly, miR-96 directly targeted a core circadian clock gene, PER2. The knockout of the miR-183/96/182 cluster in mice showed tissue-specific effects on circadian parameters and altered circadian rhythms at the behavioral level. This study identified a large number of miRNAs, including the miR-183/96/182 cluster, as circadian modulators. We provide a resource for further understanding the role of miRNAs in the circadian network and highlight the importance of miRNAs as a genome-wide layer of circadian clock regulation.

COP1 destabilizes DELLA proteins in Arabidopsis.

DELLA transcriptional regulators are central components in the control of plant growth responses to the environment. This control is considered to be mediated by changes in the metabolism of the hormones gibberellins (GAs), which promote the degradation of DELLAs. However, here we show that warm temperature or shade reduced the stability of a GA-insensitive DELLA allele in Arabidopsis thaliana Furthermore, the degradation of DELLA induced by the warmth preceded changes in GA levels and depended on the E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1). COP1 enhanced the degradation of normal and GA-insensitive DELLA alleles when coexpressed in Nicotiana benthamiana. DELLA proteins physically interacted with COP1 in yeast, mammalian, and plant cells. This interaction was enhanced by the COP1 complex partner SUPRESSOR OF phyA-105 1 (SPA1). The level of ubiquitination of DELLA was enhanced by COP1 and COP1 ubiquitinated DELLA proteins in vitro. We propose that DELLAs are destabilized not only by the canonical GA-dependent pathway but also by COP1 and that this control is relevant for growth responses to shade and warm temperature.

Phosphorylation of RNA Polymerase II by CDKC;2 Maintains the Arabidopsis Circadian Clock Period.

The circadian clock is an internal timekeeping system that governs about 24 h biological rhythms of a broad range of developmental and metabolic activities. The clocks in eukaryotes are thought to rely on lineage-specific transcriptional-translational feedback loops. However, the mechanisms underlying the basic transcriptional regulation events for clock function have not yet been fully explored. Here, through a combination of chemical biology and genetic approaches, we demonstrate that phosphorylation of RNA polymerase II by CYCLIN DEPENDENT KINASE C; 2 (CDKC;2) is required for maintaining the circadian period in Arabidopsis. Chemical screening identified BML-259, the inhibitor of mammalian CDK2/CDK5, as a compound lengthening the circadian period of Arabidopsis. Short-term BML-259 treatment resulted in decreased expression of most clock-associated genes. Development of a chemical probe followed by affinity proteomics revealed that BML-259 binds to CDKC;2. Loss-of-function mutations of cdkc;2 caused a long period phenotype. In vitro experiments demonstrated that the CDKC;2 immunocomplex phosphorylates the C-terminal domain of RNA polymerase II, and BML-259 inhibits this phosphorylation. Collectively, this study suggests that transcriptional activity maintained by CDKC;2 is required for proper period length, which is an essential feature of the circadian clock in Arabidopsis.

Isoform-selective regulation of mammalian cryptochromes.

CRY1 and CRY2 are essential components of the circadian clock controlling daily physiological rhythms. Accumulating evidences indicate distinct roles of these highly homologous proteins, in addition to redundant functions. Therefore, the development of isoform-selective compounds represents an effective approach towards understanding the similarities and differences of CRY1 and CRY2 by controlling each isoform individually. We conducted phenotypic screenings of circadian clock modulators, and identified KL101 and TH301 that selectively stabilize CRY1 and CRY2, respectively. Crystal structures of CRY-compound complexes revealed conservation of compound-binding sites between CRY1 and CRY2. We further discovered a unique mechanism underlying compound selectivity in which the disordered C-terminal region outside the pocket was required for the differential effects of KL101 and TH301 against CRY isoforms. By using these compounds, we found a new role of CRY1 and CRY2 as enhancers of brown adipocyte differentiation, providing the basis of CRY-mediated regulation of energy expenditure.

Clocks not winding down: unravelling circadian networks.

An intrinsic clock enables an organism to anticipate environmental changes and use energy sources more efficiently, thereby conferring an adaptive advantage. Having an intrinsic clock to orchestrate rhythms is also important for human health. The use of systems biology approaches has advanced our understanding of mechanistic features of circadian oscillators over the past decade. The field is now in a position to develop a multiscale view of circadian systems, from the molecular level to the intact organism, and to apply this information for the development of new therapeutic strategies or for enhancing agricultural productivity in crops.

GIGANTEA gates gibberellin signaling through stabilization of the DELLA proteins in Arabidopsis.

Circadian clock circuitry intersects with a plethora of signaling pathways to adequately time physiological processes to occur at the most appropriate time of the day and year. However, our mechanistic understanding of how the clockwork is wired to its output is limited. Here we uncover mechanistic connections between the core clock component GIGANTEA (GI) and hormone signaling through the modulation of key components of the transduction pathways. Specifically, we show how GI modulates gibberellin (GA) signaling through the stabilization of the DELLA proteins, which act as negative components in the signaling of this hormone. GI function within the GA pathway is required to precisely time the permissive gating of GA sensitivity, thereby determining the phase of GA-regulated physiological outputs.

Casein kinase 1 family regulates PRR5 and TOC1 in the Arabidopsis circadian clock.

The circadian clock provides organisms with the ability to adapt to daily and seasonal cycles. Eukaryotic clocks mostly rely on lineage-specific transcriptional-translational feedback loops (TTFLs). Posttranslational modifications are also crucial for clock functions in fungi and animals, but the posttranslational modifications that affect the plant clock are less understood. Here, using chemical biology strategies, we show that the Arabidopsis CASEIN KINASE 1 LIKE (CKL) family is involved in posttranslational modification in the plant clock. Chemical screening demonstrated that an animal CDC7/CDK9 inhibitor, PHA767491, lengthens the Arabidopsis circadian period. Affinity proteomics using a chemical probe revealed that PHA767491 binds to and inhibits multiple CKL proteins, rather than CDC7/CDK9 homologs. Simultaneous knockdown of Arabidopsis CKL-encoding genes lengthened the circadian period. CKL4 phosphorylated transcriptional repressors PSEUDO-RESPONSE REGULATOR 5 (PRR5) and TIMING OF CAB EXPRESSION 1 (TOC1) in the TTFL. PHA767491 treatment resulted in accumulation of PRR5 and TOC1, accompanied by decreasing expression of PRR5- and TOC1-target genes. A prr5 toc1 double mutant was hyposensitive to PHA767491-induced period lengthening. Together, our results reveal posttranslational modification of transcriptional repressors in plant clock TTFL by CK1 family proteins, which also modulate nonplant circadian clocks.

Control of plant stem cell function by conserved interacting transcriptional regulators.

Plant stem cells in the shoot apical meristem (SAM) and root apical meristem are necessary for postembryonic development of aboveground tissues and roots, respectively, while secondary vascular stem cells sustain vascular development. WUSCHEL (WUS), a homeodomain transcription factor expressed in the rib meristem of the Arabidopsis SAM, is a key regulatory factor controlling SAM stem cell populations, and is thought to establish the shoot stem cell niche through a feedback circuit involving the CLAVATA3 (CLV3) peptide signalling pathway. WUSCHEL-RELATED HOMEOBOX 5 (WOX5), which is specifically expressed in the root quiescent centre, defines quiescent centre identity and functions interchangeably with WUS in the control of shoot and root stem cell niches. WOX4, expressed in Arabidopsis procambial cells, defines the vascular stem cell niche. WUS/WOX family proteins are evolutionarily and functionally conserved throughout the plant kingdom and emerge as key actors in the specification and maintenance of stem cells within all meristems. However, the nature of the genetic regime in stem cell niches that centre on WOX gene function has been elusive, and molecular links underlying conserved WUS/WOX function in stem cell niches remain unknown. Here we demonstrate that the Arabidopsis HAIRY MERISTEM (HAM) family of transcription regulators act as conserved interacting cofactors with WUS/WOX proteins. HAM and WUS share common targets in vivo and their physical interaction is important in driving downstream transcriptional programs and in promoting shoot stem cell proliferation. Differences in the overlapping expression patterns of WOX and HAM family members underlie the formation of diverse stem cell niche locations, and the HAM family is essential for all of these stem cell niches. These findings establish a new framework for the control of stem cell production during plant development.

Nuclear receptor HNF4A transrepresses CLOCK:BMAL1 and modulates tissue-specific circadian networks.

Either expression level or transcriptional activity of various nuclear receptors (NRs) have been demonstrated to be under circadian control. With a few exceptions, little is known about the roles of NRs as direct regulators of the circadian circuitry. Here we show that the nuclear receptor HNF4A strongly transrepresses the transcriptional activity of the CLOCK:BMAL1 heterodimer. We define a central role for HNF4A in maintaining cell-autonomous circadian oscillations in a tissue-specific manner in liver and colon cells. Not only transcript level but also genome-wide chromosome binding of HNF4A is rhythmically regulated in the mouse liver. ChIP-seq analyses revealed cooccupancy of HNF4A and CLOCK:BMAL1 at a wide array of metabolic genes involved in lipid, glucose, and amino acid homeostasis. Taken together, we establish that HNF4A defines a feedback loop in tissue-specific mammalian oscillators and demonstrate its recruitment in the circadian regulation of metabolic pathways.

ZINC-FINGER interactions mediate transcriptional regulation of hypocotyl growth in Arabidopsis.

Integration of environmental signals and interactions among photoreceptors and transcriptional regulators is key in shaping plant development. TANDEM ZINC-FINGER PLUS3 (TZP) is an integrator of light and photoperiodic signaling that promotes flowering in Arabidopsis thaliana Here we elucidate the molecular role of TZP as a positive regulator of hypocotyl elongation. We identify an interacting partner for TZP, the transcription factor ZINC-FINGER HOMEODOMAIN 10 (ZFHD10), and characterize its function in coregulating the expression of blue-light-dependent transcriptional regulators and growth-promoting genes. By employing a genome-wide approach, we reveal that ZFHD10 and TZP coassociate with promoter targets enriched in light-regulated elements. Furthermore, using a targeted approach, we show that ZFHD10 recruits TZP to the promoters of key coregulated genes. Our findings not only unveil the mechanism of TZP action in promoting hypocotyl elongation at the transcriptional level but also assign a function to an uncharacterized member of the ZFHD transcription factor family in promoting plant growth.

Changes in ambient temperature are the prevailing cue in determining Brachypodium distachyon diurnal gene regulation.

Plants are continuously exposed to diurnal fluctuations in light and temperature, and spontaneous changes in their physical or biotic environment. The circadian clock coordinates regulation of gene expression with a 24 h period, enabling the anticipation of these events. We used RNA sequencing to characterize the Brachypodium distachyon transcriptome under light and temperature cycles, as well as under constant conditions. Approximately 3% of the transcriptome was regulated by the circadian clock, a smaller proportion than reported in most other species. For most transcripts that were rhythmic under all conditions, including many known clock genes, the period of gene expression lengthened from 24 to 27 h in the absence of external cues. To functionally characterize the cyclic transcriptome in B. distachyon, we used Gene Ontology enrichment analysis, and found several terms significantly associated with peak expression at particular times of the day. Furthermore, we identified sequence motifs enriched in the promoters of similarly phased genes, some potentially associated with transcription factors. When considering the overlap in rhythmic gene expression and specific pathway behavior, thermocycles was the prevailing cue that controlled diurnal gene regulation. Taken together, our characterization of the rhythmic B. distachyon transcriptome represents a foundational resource with implications in other grass species.

Tissue-specific clocks in Arabidopsis show asymmetric coupling.

Many organisms rely on a circadian clock system to adapt to daily and seasonal environmental changes. The mammalian circadian clock consists of a central clock in the suprachiasmatic nucleus that has tightly coupled neurons and synchronizes other clocks in peripheral tissues. Plants also have a circadian clock, but plant circadian clock function has long been assumed to be uncoupled. Only a few studies have been able to show weak, local coupling among cells. Here, by implementing two novel techniques, we have performed a comprehensive tissue-specific analysis of leaf tissues, and show that the vasculature and mesophyll clocks asymmetrically regulate each other in Arabidopsis. The circadian clock in the vasculature has characteristics distinct from other tissues, cycles robustly without environmental cues, and affects circadian clock regulation in other tissues. Furthermore, we found that vasculature-enriched genes that are rhythmically expressed are preferentially expressed in the evening, whereas rhythmic mesophyll-enriched genes tend to be expressed in the morning. Our results set the stage for a deeper understanding of how the vasculature circadian clock in plants regulates key physiological responses such as flowering time.

Arabidopsis B-BOX32 interacts with CONSTANS-LIKE3 to regulate flowering.

Plants have the ability to respond to seasonal environmental variations by monitoring day length to initiate flowering. The transition from vegetative to the reproductive stage is the critical developmental switch in flowering plants to ensure optimal fitness and/or yield. It has been previously reported that B-BOX32 (BBX32) has the potential to increase grain yield when ectopically expressed in soybean. In the present study, we performed a detailed molecular characterization of the Arabidopsis B-box domain gene BBX32 We showed that the circadian clock in Arabidopsis regulates BBX32 and expressed in the early morning. To understand the molecular mechanism of BBX32 regulation, we performed a large-scale yeast two-hybrid screen and identified CONSTANS-LIKE 3 (COL3)/BBX4 as one of its interacting protein partners. Using different genetic and biochemical assays, we have validated this interaction and shown that COL3 targets FT in the presence of BBX32 to regulate the flowering pathway. Based on these findings, we hypothesized that this BBX32-COL3 module could be an additional regulatory mechanism affecting the reproductive development in Arabidopsis that could be translated to crops for increased agricultural productivity.