CLOCK-BMAL1 transaktivasyonunu etkileyen yeni transkripsiyon faktorlerinin tanımlanması

Circadian clocks are self-sustained time-keeping systems that generate circadian rhythms with a period of approximately 24 hours. Circadian clocks are internal pacemakers that influence human physiology, endocrinology, xenobiotic detoxification, cell growth, and behavior. In mammals, the circadian clock mechanism involves several proteins that participate in positive and negative transcriptional feedback loops. Proteins involved in the positive feedback loop include BMAL1 and CLOCK proteins. These proteins form heterodimers and bind to E-box elements (CACGTG) in promoter of period (Per), cryptochrome (Cry) and other clock-controlled genes. PER and CRY proteins form heterodimers that interact with casein kinase I ε (CKIε) and then translocate into the nucleus where CRY acts as a negative regulator of BMAL1:CLOCK driven transcription. Genetic studies on mouse indicated that indeed there are more core clock components to regulate core clock at the molecular level. To identify components that have an effect on the BMAL1:CLOCK transactivation, high-throughput luciferase reporter assay was utilized to screen 1400 mammalian transcription factors. Initial screening showed that WW domain-containing transcription regulator protein 1 (WWTR1) is one of the top candidates that showed high repression activity for BMAL1:CLOCK driven transcription on Per1 promoter. Herein, I demonstrate that this repression activity is achieved by physical interaction of the WWTR1 with Bmal1 protein with co-immunoprecipitation and bi-molecular fluorescence complementation assay (BiFC). To see its effect on the circadian clock, Wwtr1 was downregulated by shRNA in NIH3T3 Per1: dluc and U2-OS Bmal1: dluc cell lines. There was damping in amplitude and advance in phase of oscillation of rhythm in both cell lines. Additionally, knockdown of Wwtr1 affected the transcriptional regulation of the core clock genes, especially transcription of the Bmal1 and Cry1 genes. Furthermore, WWTR1 appears to acts as co-activator on Cry1 and Bmal1 promoters. ChIP analysis also demonstrates WWTR1 occupation on Cry1 promoter. Collectively all these results suggest a potential new core clock component, WWTR1, for the regulation of circadian rhythms by repressing BMAL1:CLOCK transactivation and by regulating Bmal1 and Cry1 transcriptional level.