Mechanism of replisome-mediated parental histone inheritance

Abstract

Inheritance of histones from parental DNA during DNA replication is important for the maintenance of epigenetic information and chromatin assembly. Decades of studies have demonstrated that parental histones are symmetrically segregated to the two newly synthesized daughter DNA strands. However, the mechanism(s) by which parental histones are inherited in coordination with DNA replication remain unclear. In my Ph.D. thesis, I use an inducible heterochromatin domain in Schizosaccharomyces pombe as a model system to study the requirement of a replisome-associated histone chaperone network for epigenetic maintenance of heterochromatin. Using this system, I demonstrated that several previously reported replisome-associated histone binding proteins and the histone chaperone FACT are required for epigenetic inheritance of heterochromatin. In addition, I discovered that epigenetic maintenance of heterochromatin requires the full fork protection complex consisting of Swi1, Swi3 and Mrc1 proteins. In particular, I showed that the non-essential DNA checkpoint factor Mrc1 is required for epigenetic inheritance through a newly identified conserved histone binding activity. Through collaborations examining the parental histone occupancy at newly synthesized DNA, we discovered that mutations in the Mrc1 histone-binding domain disrupt parental histone transfer to both the leading and lagging DNA strands. Using AlphaFold predictions, I found that the Mrc1 histone-binding domain preferentially locates at the center of the replication fork, suggesting that Mrc1 acts as a parental histone distribution site in the replisome. With additional AlphaFold predictions, I identified multiple histone-binding domains and FACT-binding domains in the replisome. Several subcomplexes containing FACT and histones are predicted to form through these interaction interfaces. Genetic and biochemical experiments further confirmed that these newly identified interaction interfaces are required for heterochromatin maintenance. Collectively, these data led me to propose a model by which parental histones are captured by the FACT complex from the partially disassembled nucleosome induced by the replicative helicase and then inherited through multiple histone transfer intermediate sites at the replication fork. My thesis work provides a mechanistic framework for studying the dynamics of parental histone inheritance at the replication fork. Future studies are needed to validate the presence of all parental histone transfer intermediate sites at the replication fork, determine the order of parental histone movements, and investigate the role of replisome-mediated parental histone inheritance in the development of multicellular organisms.