A putative condensin loading factor that controls yeast chromosome III architecture Abstract Manikarna Dinda1, Ryan D. Fine1, Mingguang Li1,2, and Jeffrey S. Smith1 1Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908. 2Department of Laboratory Medicine, Jilin Medical University, 132013, China. Condensin plays an important and evolutionarily conserved role in mitotic chromosome condensation, three-dimensional genome organization and regulation of gene expression. Condensin is a multi-subunit complex that has natural affinity for the promoters of highly transcribed genes, and also associates with specific transcription factors. However, a general mechanism for functional loading of the complex onto chromatin remains elusive. Our lab previously demonstrated that in haploid MATa yeast cells, condensin and Sir2 (a histone deacetylase) both associate with the recombination enhancer (RE), a cis-acting element on chromosome III that directs donor preference of mating-type switching. Here, Sir2 locally regulates transcription of a small gene called RDT1, while condensin contributes to the 3-dimensional organization of chromosome III, as well as donor preference, indicating that the RDT1 promoter region acts as a locus control region (Li et al., 2019). We have now further characterized the mechanism of condensin recruitment to the RDT1 promoter LCR, and uncovered a critical role for a non-meiotic version of the monopolin complex, known as cohibin (Lrs4 and Csm1 subunits), analogous to its known role in recruiting condensin to the rDNA locus. To test if cohibin functions more generally in condensin loading, or as a condensin accessory factor, we have performed ChIP-seq for genome-wide condensin binding sites in WT and lrs4∆ strains. Numerous Brn1 peaks (including RDT1 and the rDNA) were eliminated or significantly reduced by lrs4∆, consistent with the condensin loader hypothesis. Micro-C XL was then used to characterize the general effects of defective condensin recruitment on genomic conformation in a lrs4∆mutant, or when the Brn1 condensin subunit was depleted using an auxin-inducible degron system. Alterations were observed on multiple chromosomes, though the most severe changes occurred on chromosome III, which had significant negative effects on the efficiency of mating-type switching, as well as donor preference. We therefore hypothesize that cohibin (Lrs4/Csm1) is indeed a condensin loader at the RDT1 promoter region, where it establishes chromosome III conformation, potentially through a loop extrusion mechanism. Evidence for direct recruitment of condensin by the cohibin complex at other genomic sites and any possible role for cohibin in chromosome condensation are currently under investigation. Dr. Chongzhi Zang, University of Virginia Dr. Zhenjia Wang, University of Virginia Supported by NIH GM075240 and GM127394 Aim Establish whether Lrs4 is a condensin loader at RDT1 promoter region RDT1 promoter region HIS4 promoter region 6. lrs4 Δ reduces condensin binding at the RDT1 promoter region Condensin Sir2 RDT1 Mcm1 minimum 700bp RE Mcm1 ARS304 100bp HML HMR MAT RDT1 promoter region CEN Condensin 11. Hypothetical loop extrusion model after binding of condensin to the RDT1 promoter • Lrs4 is the condensin loader at RDT1 promoter region. • Condensin is recruited to the RDT1 promoter and regulates mating type switching by maintaining the chromosome conformation. • Presumably, condensin by loop extrusion is facilitating the interaction of HML with MAT to repair DNA breaks in MAT a cells. 12. Conclusion 5. Proposed model of condensin and Sir2 recuitment to the RDT1 promoter by Lrs4 and Mcm1 1. Mechanism of mating type switching in budding yeast 10. Condensin depletion leads to loss of interaction between MATa-HMLα, HMLα- HMRa in ChrIII during mating type switching 7. Lrs4 helps to recruit condensin at RDT1 promoter in MATa cells MAT a cells MAT ! cells 4. A putative