2018 Winner: The regulatory roles of alternative splicing and intrinsically disordered regions of proteins in circadian rhythms

Project Information
The regulatory roles of alternative splicing and intrinsically disordered regions of proteins in circadian rhythms
Physical and Biological Sciences
The main goal for my independent research project was to gain insight how circadian rhythms are regulated at a molecular level. Most researchers in the field of chronobiology take genetic and cellular approaches, which leads to a deficit in knowledge of the detailed molecular mechanisms of circadian regulation. Although the founders of chronobiology, Colin Pittendrigh and Jurgen Aschoff, made many important findings in the 1950s-1960s, we still do not fully understand how circadian rhythms are regulated at the molecular level (Aschoff, 1965; Pittendrigh and Minis, 1964). While we understand the genetic network that comprises the clock and how the clock genes work with one another, we do not have enough insight into the biochemical mechanisms of the clock to understand why mutation in clock genes elicit particular effects and are linked to certain disorders. In order to understand the regulation of circadian rhythms at the biochemical level, we need to study protein-protein interactions between the core circadian proteins: the heterodimer transcription factor CLOCK:BMAL1, and their repressors (Gustafson and Partch, 2015). The first repressor I studied was the Clock-Interacting Protein Circadian, CIPC. This repressor was discovered using a two-hybrid assay that tested for protein interaction between CLOCK WT and CLOCKΔ19 (Zhao et al., 2007). CLOCKΔ19 is a mutant allele through alternative splicing that leads to an internal deletion of 51 residues encoded by Exon 19 in the disordered C-terminus of CLOCK. CIPC does not have any known vertebrate homologs and its only known role is to inhibit transcriptional activation by CLOCK:BMAL1(Zhao et al., 2007). CIPC loses the ability to inhibit CLOCK:BMAL1 with the deletion of CLOCK Exon 19. In addition to studying the Exon 19-CIPC interaction, I studied the role of dimerization of Exon 19 and how it mediates the binding of two distinct CLOCK:BMAL1 heterodimers to tandem DNA E-box promoter regions through positive cooperativity. Positive cooperativity as a result of binding to tandem sites on DNA has been shown to produce robust circadian rhythms (Shimomura et al., 2013). The second circadian repressor I studied is Cryptochrome 1, (CRY1). For this repressor, I mostly focused on the C-terminal disordered tail domain of CRY1. A single nucleotide mutation of the CRY1 gene, leads to alternative splicing and deletion of Exon 11 in CRY1 (Patke, A. et al., 2017). Patke, A. et al., had shown through cell-based assays that deleting Exon 11, a 24-residue region on the CRY1 disordered C-terminus, increases inhibition of CLOCK:BMAL1 by CRY1. This mutation is common in people with Delayed Sleep Phase Disorder (DSPD), which has an estimated prevalence of up to 1 in 75 people in the general population. We hypothesized that Exon 11 might be interfering with repression by regulating CLOCK:BMAL1 binding. The Partch Lab had previously demonstrated how CRY1, without the disordered C-terminus, interacts with CLOCK. To probe how CRY1 C-terminus might alter the interaction with CLOCK and change affinity, first we needed to determine how the disordered c-terminus interacts with CRY1 PHR and then test how it might affect the binding to CLOCK.
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  • Ivette Perez (Crown)