Although the effort to catalog the full range of epigenetic marks and assign them functional roles has been a remarkable success to date, how epigenetic states persist through replication and mitosis (epigenetic inheritance) remain as important open questions. Current theories suggest that marked histones remain associated with DNA during chromosomal replication and mitotic condensation and then serve as templates for reassembly of the interphase chromatin state. In the case of DNA replication, the requirement for DNA strands to be unwound and accessed by polymerases places severe constraints on potential mechanisms for persistence of epigenetic marks. Recent data have implicated histone chaperones in concert with chromatin remodelers and DNA polymerase complexes, as the mediators of nucleosome dissasembly and reassembly at sites of replication. However, much needs to be learned about how recycled and/or newly synthesized histones are deposited and remodeled to adopt the correct patterns of histone types and modifications. We are interested in dissecting the inheritance of epigenetic marks by exploiting proteomic approaches to analyze maturation and dynamics of replicated chromatin during S phase and mitosis. As such, we will develop methods to obtain marked chromatin via pulse chase analysis to isolate sites of DNA synthesis or chromatin at different intervals after replication. We will particularly focus on the inheritance of heterochromatin. Then, we will use quantitative proteomic analysis to follow the time-course of changes in chromatin proteins and modifications following replication, through mitosis and into the next cell cycle. Finally, we hope to determine which proteins and modifications are required for inheritance of chromatin states through replication and persistence of chromatin marks after mitosis.
We propose to better understand epigenetic inheritance by documenting the molecular events that allow newly replicated or repaired chromatin to return to its former state. Discovery of the specific mechanisms that allow patterns of expression to be passed on during normal cell division will have broad applications in understanding and treating diseases of development and aging and may identify new targets for cancer therapy.
