Our Unit is interested in the molecular mechanisms underlying regulated patterns of gene expression in circadian time frames. The circadian field provides excellent examples of the types of gaps that hypothesis-driven research is best suited to fill. After having enormous success in describing the inner workings of the biological clock, the field of molecular chronobiology is rapidly shifting its attention to the visualization of circadian changes in transcription on a global scale. But in the midst of rapidly growing databases and behind the remarkable progress in our understanding of how molecular clocks work, several important questions remain unanswered. For example, we now know that the interaction between the BMAL/CLOCK protein complex and a cis-acting element known as the E-Box lies at the interphase between the master clock machinery and a specific set of clock and clock-controlled genes. In spite of the central nature of this interaction however, no current models can explain how such a promiscuous DNA regulatory site can steer the transcriptional machinery specifically onto the promoters of circadian genes. The long-term goal of the Unit on Temporal Gene Expression is to identify the mechanisms controlling the specificity of gene expression in the circadian system.
Our specific aim i s to identify DNA sites and protein factors that function to achieve a) circadian target selection (which gene to activate and where?) and b) precise amplitude modulation (by how much?). We hypothesize the existence of yet unidentified or unrecognized regulatory factors that confer circadian value onto an E-Box. Our rationale for conducting this research is that identification of such factors is bound to reveal novel and critical pathways that modulate the location, phase and amplitude of CCG expression. To test this hypothesis the UTGE research focuses on two parallel systems where careful orchestration of gene expression is evident. The first system is the interaction between the BMAL/CLOCK transcription factor and the circadian E-Box. Our approach involves the systematic dissection of the DNA context around a subset of perfect E-Boxes known to be responsive or refractory to the input from the clock. We latch onto novel sequences with the potential to specifically modify BMAL/CLOCK action and investigate the biology of the cognate binding factors. The second system pertains to the Fos Related Antigen (Fra)-2 response, particularly in the pineal gland. The robust circadian induction of the Fra-2 gene in this slave oscillator takes place during a massive reprogramming of gene expression. We study this response through the generation of transgenic rats that express a pineal-specific dominant negative form of the Fra-2 protein. We then evaluate the role of a Fra-2 containing AP-1 factor in temporal target selection by transcriptome analysis. As a result of our research in these two areas we became particularly interested in the possibility of a cross-talk between the AP-1 family of transcription factors and the modulation of clock gene expression by the BMAL/CLOCK complex in transcriptionally oscillatory systems. Our most recent accomplishments include: 1) the in vivo validation of the differential use of a circadian E-Box in retina vs. pineal. 2) identification of Fra-2-modulated targets in the rat pineal gland. 3) the discovery of a novel cis-acting element (CTRR) that affects BMAL/CLOCK transactivation and that displays rhythmic binding in the pineal gland. 4) the discovery of a modulatory role of the Fra-2/JunD complex on the BMAL/CLOCK-dependent pathway. 5) discovering that the strongly circadian Cold inducible glycoprotein 30 gene is also a sexually dimorphic gene 5) the discovery of an acutely resettable circadian rhythm in the proteolytic activation of Sterol regulatory element binding protein (SREBP)-1 in the rodent liver. Significance The research efforts outlined above have already generated new insight into several areas, contributed to a deeper understanding of the subtle mechanisms that control circadian transcription, and provided exciting new leads for future projects. By systematically dissecting tightly controlled and very robust regulatory systems in space (AA-NAT, Fra-2, SREBP) and time (AVP, Fra-2, SREBP) we expect to find novel mechanisms for the control of gene expression. Their relevance would likely spill over into fields unrelated to chronobiology.
