Precise timing of CONSTANS (CO) gene expression is necessary for day-length discrimination for photoperiodic flowering. The FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) and GIGANTEA (GI) proteins regulate CO transcription in Arabidopsis. We demonstrate that FKF1and GI proteins form a complex in a blue-light dependent manner. The timing of this interaction regulates the timing of daytime CO expression. FKF1 function is dependent on GI, which interacts with a CO repressor, CYCLING DOF FACTOR 1 (CDF1), and controls CDF1 stability. GI, FKF1, and CDF1 proteins associate with CO chromatin. Thus, the FKF1-GI complex forms on the CO promoter in late afternoon to regulate CO expression, providing a mechanistic view of how the coincidence of light with circadian timing regulates photoperiodic flowering.Many plants monitor seasonal changes in day-length to regulate flowering time for successful reproduction (1). In Arabidopsis, regulation of daytime CO expression is the primary process of time measurement in the photoperiodic flowering pathway (2, 3). FKF1 and GI proteins positively regulate CO transcription (4, 5). FKF1 and GI gene expression has similar diurnal patterns (5, 6), implying that these proteins may interact to regulate CO.We tested their direct interaction in yeast and found that FKF1 interacts with GI (Fig. 1A). Our results obtained using truncated FKF1 proteins suggests that this interaction occurs through the FKF1 LOV (Light, Oxygen, or Voltage) domain (Fig. 1A). In addition, the GI N-terminus was sufficient to interact with FKF1 ( fig. S1).To assess whether this interaction occurs in vivo, and whether it is modulated by photoperiod or light conditions, we generated transgenic plants constitutively expressing both haemagglutinin (HA)-tagged FKF1 (HA-FKF1) and tandem affinity purification (TAP)-tagged GI (GI-TAP) proteins [35S∷HA-FKF1 35S∷GI-TAP lines (7)] for coimmunoprecipitation experiments. In the 35S∷HA-FKF1 35S∷GI-TAP #18 / fkf1 line, a similar amount of GI-TAP protein was precipitated at every time point in both long-day (16 hours light / 8 hours dark) and short-day (8 hours light / 16 hours dark) conditions (Fig. 1, B and C). HA-FKF1 protein was coimmunoprecipitated with GI-TAP protein (Fig. 1, B and C), demonstrating that GI-TAP and HA-FKF1 proteins form a complex in vivo. In both daylength conditions, the amount of coimmunoprecipitated HA-FKF1 protein increased until 4 hours after light onset, remained constant for the rest of day, and declined in the dark (Fig. 1, B and C), suggesting that light or the circadian clock modulate the FKF1 and GI interaction.We therefore analyzed the interaction in dark-grown samples. A minimal amount of HA-FKF1 was coimmunoprecipitated with GI-TAP protein in the dark (Fig. 1D) Next we analyzed how light quality (wavelength) affects this interaction. Similar amounts of FKF1 and GI interacted in blue-light irradiated samples (Fig. 1E) compared to white-light grown samples, but little interaction was observed in red-light irradiated samples ( Fig. 1E), indicati...

The circadian clock is required for adaptive responses to daily and seasonal changes in environmental conditions1-3. Light and the circadian clock interact to consolidate the phase of hypocotyl cell elongation to dawn under diurnal cycles in Arabidopsis thaliana4-7. Here we identify a protein complex (Evening Complex) composed of EARLY FLOWERING 3 (ELF3), EARLY FLOWERING 4 (ELF4) and the transcription factor LUX ARRHYTHMO (LUX) that directly regulates plant growth8-12. ELF3 is both necessary and sufficient to form a complex between ELF4 and LUX, and the complex is diurnally regulated, peaking at dusk. ELF3, ELF4 and LUX are required for the proper expression of the growth-promoting transcription factors PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) and PIF5 under diurnal conditions4,6,13. LUX targets the complex to the promoters of PIF4 and PIF5 in vivo. Mutations in PIF4 and/or PIF5 are epistatic to the loss of the ELF4-ELF3-LUX complex, suggesting that regulation of PIF4 and PIF5 is a critical function of the complex. Therefore, the Evening Complex underlies the molecular basis for circadian gating of hypocotyl growth in the early evening.

During fasting, mammals maintain glucose homeostasis by stimulating hepatic gluconeogenesis1. Elevations in circulating glucagon (GLU) and epinephrine trigger the cAMP mediated phosphorylation of Creb and dephosphorylation of the Creb coactivator Crtc22. Although the underlying mechanism is unclear, hepatic gluconeogenesis is also regulated by the circadian clock, which coordinates glucose metabolism with changes in the external environment3–6. Here we show that Creb activity during fasting is modulated by Cryptochromes (Cry1 and Cry2), core components of the clock that are rhythmically expressed in the liver. Cry was elevated during the night/day transition, when it reduced fasting gluconeogenic gene expression by blocking GLU-mediated increases in intracellular cAMP concentrations and in the PKA-mediated phosphorylation of Creb. In biochemical reconstitution studies, we found that Cry inhibited accumulation of cAMP in response to G protein coupled receptor (GPCR) activation but not to forskolin, a direct activator of adenyl cyclase. Cry appeared to modulate GPCR activity directly through interaction with Gsα . As hepatic over-expression of Cry lowered blood glucose concentrations and improved insulin sensitivity in insulin resistant db/db mice, our results suggest that compounds which enhance Cry activity may provide therapeutic benefit to individuals with type II diabetes.

Dosage compensation in mammals is achieved by the transcriptional inactivation of one X chromosome in female cells. From the time X chromosome inactivation was initially described, it was clear that several mechanisms must be precisely integrated to achieve correct regulation of this complex process. X-inactivation appears to be triggered upon differentiation, suggesting its regulation by developmental cues. Whereas any number of X chromosomes greater than one is silenced, only one X chromosome remains active. Silencing on the inactive X chromosome coincides with the acquisition of a multitude of chromatin modifications, resulting in the formation of extraordinarily stable facultative heterochromatin that is faithfully propagated through subsequent cell divisions. The integration of all these processes requires a region of the X chromosome known as the X-inactivation center, which contains the Xist gene and its cis-regulatory elements. Xist encodes an RNA molecule that plays critical roles in the choice of which X chromosome remains active, and in the initial spread and establishment of silencing on the inactive X chromosome. We are now on the threshold of discovering the factors that regulate and interact with Xist to control X-inactivation, and closer to an understanding of the molecular mechanisms that underlie this complex process.

Summary Circadian clocks provide an adaptive advantage by allowing organisms to anticipate daily and seasonal environmental changes [1, 2]. Eukaryotic oscillators rely on complex hierarchical networks composed of transcriptional and post-translational regulatory circuits [3]. In Arabidopsis, current representations of the circadian clock consist of three or four interlocked transcriptional feedback loops [3, 4]. Although molecular components contributing to different domains of these circuits have been described, how the loops are connected at the molecular level is not fully understood. Genetic screens previously identified LUX ARRHYTHMO (LUX) [5], also known as PHYTOCLOCK1 (PCL1) [6], an evening-expressed putative transcription factor essential for circadian rhythmicity. We determined the in vitro DNA-binding specificity for LUX by using universal protein binding microarrays; we then demonstrated that LUX directly regulates the expression of PSEUDO RESPONSE REGULATOR9 (PRR9), a major component of the morning transcriptional feedback circuit, through association with the newly discovered DNA-binding site. We also show that LUX binds to its own promoter, defining a new negative autoregulatory feedback loop within the core clock. These novel connections between the archetypal loops of the Arabidopsis clock represent a significant advance towards defining the molecular dynamics underlying the circadian network in plants and provide the first mechanistic insight into the molecular function of the previously orphan clock factor LUX.