Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Session: 2
Room: Exhibit Hall
Katherine Duseau - University of New Haven
Co-Author(s): Dr. Kevin Li, Yale University, New Haven, CT
Chromosomal contacts play a role in many cellular functions such as gene regulation, DNA replication, and DNA repair. The frequency of interactions between different genomic loci is influenced by the organization of the chromatin in the nucleus. Chromatin confirmation capture techniques, such as Hi-C, have revealed the existence of higher order genome structures such as cohesin- mediated extruded loops called topologically associated domains (TADs) and liquid-liquid phase separated euchromatic and heterochromatic compartments. The dynamics of chromatin interactions across these structures is largely unknown. This study aims to evaluate how genomic organization affects chromosomal contacts in the budding yeast, S. cerevisiae. We measured the first passage time, the time required for two loci on a single chromosome to come in contact, by using a Cre-lox assay which comprises of Promoter-loxP and loxP-EGFP sites integrated at predetermined regions of the genome. The controlled induction of a Cre-recombinase will drive a recombination event when two loxP sites interact, resulting in the expression of GFP, which acts as reporter single for true contact between the inserted loxP sites. Imaging cells over time by fluorescence microscopy allows for the measurement of the first passage time between predetermined loci that was previously not possible with existing experimental methods. We have performed proof of concept experiments that show that the Cre-lox assay can detect first passage times in S. cerevisiae. Further experimentation will better assess the first passage time in both S. cerevisiae and S. pombe for numerous genomic separations of loxP inserts and in the absence or presence of TADs and compartments to elucidate the role of higher order structures on chromatin dynamics.
Funder Acknowledgement(s): Funding: YALE University PEBNSF EFMA1830904
Faculty Advisor: Dr. Megan King, megan.king@yale.edu
Role: My role in this research was to assist in the proof-of-concept experiments. In this aspect of the project, I trained to use fluorescent microscopes and analyze images. Additionally, I worked to design and integrate new lox-p sights in S. cerevisiae. Specifically, I designed primers for the new sights of integration, ran PCRs to amplify lox-p cassettes with the newly designed primers, and performed transformations in S. cerevisiae.