Circadian chromatin landscape and identification of novel transcriptional regulators of the circadian clock

• Applicant: Professor Dr. Achim Kramer
• Subject Area: General Genetics and Functional Genome Biology ; Biochemistry ; Cell Biology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 532624261

Circadian (~24 hr) clocks are molecular oscillators present in essentially all mammalian cells and drive daily changes in physiology, metabolism and behavior. Misaligned or disrupted clocks are common in modern society and are associated with many widespread diseases, such as metabolic syndromes, cancer, psychiatric disorders and cardiovascular pathologies. Thus, exploring the mechanisms that establish coherent, large amplitude and correctly phased circadian rhythms in mammalian cells is crucial for understanding the pathophysiological mechanisms associated with dysfunctional circadian clocks. At the center of the molecular oscillator is the circadian regulation of gene transcription. Previous studies have identified clock protein complexes interacting with the transcriptional machinery to drive circadian rhythms of transcription. Primarily due to technological limitations, these studies focused on a few regulators and left many gaps in our understanding of circadian transcription dynamics. With recent advances in quantitative genomic locus-specific proteomics, there is now an opportunity to overcome these limitations. In the preliminary work presented, we successfully adapted and validated a novel genomic locus proteomics method to study the time-of-day dependent change in protein binding to a key chromatin region of circadian transcription (i.e. the E-box). Briefly, catalytically inactive RNA-guided nuclease CAS9 (dCAS9) is fused to ascorbate peroxidase APEX2. Single guide RNAs target this complex targets a specific locus in the genome where APEX2 is activated to biotinylate proteins in its proximity. Biotinylated proteins are purified and analyzed via mass spectrometry. Using this new technology, our goal is (i) to provide the first comprehensive and unbiased characterization of circadian proteins regulating circadian gene expression; and (ii) to identify novel transcriptional regulators critical to circadian dynamics. Of particular interest are those regulators that link the circadian machinery to other important cellular mechanisms (e.g. cell cycle, cell-cell communication, etc.). Overall, this project will provide new information on the mechanism of circadian oscillator function, thus laying the groundwork for the identification of new targets for the development of better treatment strategies for circadian clock-related disorders.

• DFG Programme: Research Grants