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How to Replicate Chromatin - Maturation, Timing Control and Stress-Induced Aberrations

Final Report Summary - CHROMATINREPLICATION (How to Replicate Chromatin - Maturation, Timing Control and Stress-Induced Aberrations)

Generally, all cells in the human body contains the same genomic information. However, different cell types have different properties and serve different functions in the body. So-called epigenetic mechanisms are involved in the establishment and maintenance of the different cell types during development. Chromatin, the structure that organizes the DNA within the nucleus, contributes to such epigenetic cell memory. Thus, when a cell divides, both the DNA and its organization into chromatin must be duplicated in order for the daughter cells to reproduce the identity of the mother cell. While we understand how DNA is replicated, very little is known about how the chromatin landscape is reproduced when cells divide. In this project, we have developed a new technology, termed nascent chromatin capture (NCC), that allowed us to investigate the complex process of chromatin replication using large-scale proteomics (Alabert et al., 2014 Nature Cell Biol). Chromatin is composed of DNA and thousands of proteins, including nucleosomes consisting of DNA wrapped around a core of histone proteins. Histones can be chemically modified in cell type specific patterns. Using NCC, we have found that chromatin replication leads to dramatic changes in hundreds of proteins and provided an overview of how chromatin is gradually re-build on the new DNA stands. This analysis also identified new proteins involved in chromatin replication. We then further developed NCC to track the chemical modifications on histones during DNA replication (Alabert, Bath et al., 2015 Genes Dev). We knew that chromatin is re-build from old recycled histones along with newly synthesized ones. Our work showed that the chemical modifications are maintained on the old histone during recycling, which allows the cell type specific modification pattern to be copied during DNA replication. This is thus one important mechanism for maintaining cell identity in dividing cells. In addition, the new histones must acquire marks similar to the old ones. Measuring the in vivo kinetics for establishment of histone modifications, we found the level of key repressive histone modifications is continuously oscillating in dividing cells reaching a minimum after DNA replication and then building up until the next round of replication, with potential implications for cell fate decisions.
Using NCC to address how genotoxic insults impact on chromatin replication, we have obtained a comprehensive view of the dramatic changes in protein composition that occur upon replication stress. We discovered a fundamental mechanism that allow cells to determine whether a region of the genome has been replicated or not (Saredi, Huang et al., 2016 Nature). This is important because DNA lesions in replicated regions can be repaired by so-called error-free mechanisms that utilize the sister DNA copy as a blueprint for repair. Our analysis identified a specific histone modification, or rather the absence of a modification at histone H4 lysine 20, as a signature of replicated genomic regions. This signature is present from the time of replication, due to deposition of new unmarked histones, until mitosis where the sister chromosomes segregate to daughter cells. Importantly, we identified a reader protein complex, called TONSL-MMS22L, which directly recognizes the mark (histone H4 unmodified at lysine 20). This complex is involved in error-free DNA repair, and thus via its histone reader function integrates information about the replication status of a loci into the choice of repair pathway. This has important implications for our understanding of genome maintenance mechanisms and opens new avenues for targeting error-free DNA repair in cancer treatment.