Project Summary Expansions of simple DNA repeats cause over 30 heritable neuromuscular and neurodegenerative disorders. For example, the two major types of myotonic dystrophy are repeat expansion disorders. DM1 is caused by long CTG trinucleotide repeats in the 5’ UTR of DMPK whereas DM2 is caused by long tetranucleotide CCTG repeats in an intron of ZNF9. Experimental systems from bacteria to Drosophila to human cells have been established to investigate the mechanisms by which CTG sequences expand, or increase in length. Such studies have implicated specific roles of DNA replication, DNA repair, and transcription in the expansion process. In contrast, CCTG repeats are more poorly understood, and it is unclear whether the same molecular mechanisms contributing to CTG repeat expansions also apply to CCTG repeats. This is a pressing question since individuals with DM2 have, on average, 5000 CCTG repeats in ZNF9. Moreover, the incidence of such long repeat lengths make delineating the mechanism of repeat contraction particularly attractive as a therapeutic prospect. Thus, the work proposed here seeks to establish budding yeast Saccharomyces cerevisiae as a robust experimental system to investigate CCTG repeat expansions and contractions. Specifically, the proposed research will investigate the genetic control of CCTG repeat instability and define the proteins and chromatin modifications associated with CCTG repeats. My overarching hypothesis is that the genes involved in CCTG repeat instability may partially overlap with those of CTG repeats, but there will be a unique set of players owing to the distinct effect of CCTG repeats on DNA secondary structure, chromatin state, and replication dynamics. Towards this end, I will determine the effect of CCTG repeat length, orientation, and transcription on expansions and contractions. I will identify genetic and molecular determinants of CCTG repeat instability using both candidate and unbiased genetic screening approaches. In particular, identifying genes involved in CCTG repeat contraction through an unbiased genetic screen will potentially uncover genes that have no previously known role in DNA repeat maintenance. Finally, I will define the protein and chromatin landscape at CCTG repeats and compare it to CTG repeats using the innovative CRISPR-based Chromatin Affinity Purification with Mass Spectrometry (CRISPR-ChAP-MS) approach. Overall, the proposed research will have a major impact on our understanding of the molecular biology of CCTG repeats, which has significant implications for human health and disease. This work will identify potential targets for preventative and therapeutic interventions for DM2 and also serve as a model for understanding the replication and maintenance of microsatellite repeats responsible for other repeat expansion disorders.