The circadian clock-associated 1 (CCA1) gene encodes a Myb-related transcription factor that has been shown to be involved in the phytochrome regulation of Lhcb1*3 gene expression and in the function of the circadian oscillator in Arabidopsis thaliana. By using a yeast interaction screen to identify proteins that interact with CCA1, we have isolated a cDNA clone encoding a regulatory (␤) subunit of the protein kinase CK2 and have designated it as CKB3. CKB3 is the only reported example of a third ␤-subunit of CK2 found in any organism. CKB3 interacts specifically with CCA1 both in a yeast two-hybrid system and in an in vitro interaction assay. Other subunits of CK2 also show an interaction with CCA1 in vitro. CK2 ␤-subunits stimulate binding of CCA1 to the CCA1 binding site on the Lhcb1*3 gene promoter, and recombinant CK2 is able to phosphorylate CCA1 in vitro. Furthermore, Arabidopsis plant extracts contain a CK2-like activity that affects the formation of a DNA-protein complex containing CCA1. These results suggest that CK2 can modulate CCA1 activity both by direct interaction and by phosphorylation of the CCA1 protein and that CK2 may play a role in the function of CCA1 in vivo.

A wide range of processes in plants, including expression of certain genes, is regulated by endogenous circadian rhythms. The circadian clock-associated 1 (CCA1) and the late elongated hypocotyl (LHY) proteins have been shown to be closely associated with clock function in Arabidopsis thaliana. The protein kinase CK2 can interact with and phosphorylate CCA1, but its role in the regulation of the circadian clock remains unknown. Here we show that plants overexpressing CKB3, a regulatory subunit of CK2, display increased CK2 activity and shorter periods of rhythmic expression of CCA1 and LHY. CK2 is also able to interact with and phosphorylate LHY in vitro. Additionally, overexpression of CKB3 shortened the periods of four known circadian clock-controlled genes with different phase angles, demonstrating that many clock outputs are affected. This overexpression also reduced phytochrome induction of an Lhcb gene. Finally, we found that the photoperiodic flowering response, which is influenced by circadian rhythms, was diminished in the transgenic lines, and that the plants flowered earlier on both long-day and short-day photoperiods. These data demonstrate that CK2 is involved in regulation of the circadian clock in Arabidopsis. C ircadian rhythms are driven by endogenous biological clocks that regulate many biochemical, physiological, and behavioral processes in a wide variety of organisms. According to current models, circadian clocks regulating these rhythms consist of input pathways, a central oscillator, and output pathways (1-3). Oscillators are thought to generate rhythms by a transcription-translation negative feedback loop (4-8). Studies in cyanobacteria, Neurospora, Drosophila, and mouse have found that both positive and negative elements that activate and inhibit the transcription of clock genes are required to maintain the loop. In addition, posttranscriptional and posttranslational regulation play important roles in circadian clocks in Drosophila and Neurospora (7-10). The oscillator can be entrained by input pathways from environmental cues such as light and temperature, and, in turn, regulates specific cellular events such as expression of clock-controlled genes (1-3).Until recently, little was known about the molecular mechanisms of circadian clocks in plants (11-13). In Arabidopsis thaliana, the toc1 mutation affects the period of many circadian rhythms (14,15). Although the corresponding gene has not yet been cloned, it is thought that TOC1 encodes a component of the oscillator. The ELF3 gene is proposed to act in the input pathway (16). Two Myb-related genes, circadian clock-associated 1 (CCA1) and late elongated hypocotyl (LHY), have also been identified as potential clock genes (17, 18), and CCA1 was found to act as a transcription factor for Lhcb gene expression (19). Expression of CCA1 and LHY oscillates with a circadian rhythm. Constitutive expression of CCA1 was shown to abolish several distinct circadian rhythms, suppress its own expression as well as that of LHY, and delay flowering substa...

The circadian clock is an endogenous mechanism that coordinates biological processes with daily and seasonal changes in the environment. Heterodimerization of central clock components is an important way of controlling clock function in several different circadian systems. CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYL (LHY) are Myb-related proteins that function in or close to the central oscillator in Arabidopsis (Arabidopsis thaliana). Single mutants of cca1 and lhy have a phenotype of short-period rhythms. cca1 lhy double mutants show an even shorter period phenotype than the cca1 single mutant, suggesting that CCA1 and LHY are only partially functionally redundant. To determine whether CCA1 and LHY act in parallel or synergistically in the circadian clock, we examined their expression in both light-grown and etiolated seedlings. We have shown that LHY and CCA1 bind to the same region of the promoter of a Light-harvesting chlorophyll a/b protein (Lhcb, also known as CAB). CCA1 and LHY can form homodimers, and they also colocalize in the nucleus and heterodimerize in vitro and in vivo. In Arabidopsis, CCA1 and LHY physically interact in a manner independent of photoperiod. Moreover, results from gel filtration chromatography indicate that CCA1 and LHY are present in the same large complex in plants. Taken together, these results imply that CCA1 and LHY function synergistically in regulating circadian rhythms of Arabidopsis.

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The immense growth of MEDLINE coupled with the realization that a vast amount of biomedical knowledge is recorded in free-text format, has led to the appearance of a large number of literature mining techniques aiming to extract biomedical terms and their inter-relations from the scientific literature. Ontologies have been extensively utilized in the biomedical domain either as controlled vocabularies or to provide the framework for mapping relations between concepts in biology and medicine. Literature-based approaches and ontologies have been used in the past for the purpose of hypothesis generation in connection with drug discovery. Here, we review the application of literature mining and ontology modeling and traversal to the area of drug repurposing (DR). In recent years, DR has emerged as a noteworthy alternative to the traditional drug development process, in response to the decreased productivity of the biopharmaceutical industry. Thus, systematic approaches to DR have been developed, involving a variety of in silico, genomic and high-throughput screening technologies. Attempts to integrate literature mining with other types of data arising from the use of these technologies as well as visualization tools assisting in the discovery of novel associations between existing drugs and new indications will also be presented.

The circadian clock regulates the expression of an array of Arabidopsis genes such as those encoding the LIGHT-HARVESTING CHLOROPHYLL A/B (Lhcb) proteins. We have previously studied the promoters of two of these Arabidopsis genes--Lhcb1*1 and Lhcb1*3--and identified a sequence that binds the clock protein CIRCADIAN CLOCK ASSOCIATED 1 (CCA1). This sequence, designated CCA1-binding site (CBS), is necessary for phytochrome and circadian responsiveness of these genes. In close proximity to this sequence, there exists a G-box core element that has been shown to bind the bZIP transcription factor HY5 in other light-regulated plant promoters. In the present study, we examined the importance of the interaction of transcription factors binding the CBS and the G-box core element in the control of normal circadian rhythmic expression of Lhcb genes. Our results show that HY5 is able to specifically bind the G-box element in the Lhcb promoters and that CCA1 can alter the binding activity of HY5. We further show that CCA1 and HY5 can physically interact and that they can act synergistically on transcription in a yeast reporter gene assay. An absence of HY5 leads to a shorter period of Lhcb1*1 circadian expression but does not affect the circadian expression of CATALASE3 (CAT3), whose promoter lacks a G-box element. Our results suggest that interaction of the HY5 and CCA1 proteins on Lhcb promoters is necessary for normal circadian expression of the Lhcb genes.

Drug repurposing is the process of using existing drugs in indications other than the ones they were originally designed for. It is an area of significant recent activity due to the mounting costs of traditional drug development and scarcity of new chemical entities brought to the market by bio-pharmaceutical companies. By selecting drugs that already satisfy basic toxicity, ADME and related criteria, drug repurposing promises to deliver significant value at reduced cost and in dramatically shorter time frames than is normally the case for the drug development process. The same process that results in drug repurposing can also be used for the prediction of adverse events of known or novel drugs. The analytics method is based on the description of the mechanism of action of a drug, which is then compared to the molecular mechanisms underlying all known adverse events. This review will focus on those approaches to drug repurposing and adverse event prediction that are based on the biomedical literature. Such approaches typically begin with an analysis of the literature and aim to reveal indirect relationships among seemingly unconnected biomedical entities such as genes, signaling pathways, physiological processes, and diseases. Networks of associations of these entities allow the uncovering of the molecular mechanisms underlying a disease, better understanding of the biological effects of a drug and the evaluation of its benefit/risk profile. In silico results can be tested in relevant cellular and animal models and, eventually, in clinical trials.

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