Structural Insights into the Mechanisms Controlling the Modification and the Readout of Histone H3.1

Abstract

The histone H3.1 is the canonical histone H3 inserted into new nucleosomes during replication. The unstructured N-terminal region (also referred to as the tail) of histone H3.1 is decorated by many post-translational modifications (PTMs). These PTMs serve as epigenetic signals coordinating essential nuclear processes such as transcription, replication and DNA damage repair mechanisms. These marks can be reversibly deposited by specialized "writer" domains in chromatin remodelling enzymes. Similarly, histone PTMs are recognized by "reader" domains which aid in coordinating appropriate responses and recruitment of relevant factors. The contribution of PTMs deposited on histone H3.1 to nuclear transactions has been extensively described in chromatin structure, regulation of gene expression, cell cycle progression, apoptosis, and DNA damage repair. However, with several DNA damage repair pathways occurring during different cell cycle phases, the contribution of histone H3 variants and PTMs is not fully understood. In this work, we characterized the tetratricopeptide repeat (TPR) domain of TONSOKU (TSK), a DNA damage response protein, as a variant-specific reader for histone H3.1 and established a role for H3.1 in replication-coupled DNA repair via the homologous recombination pathway. Using X-ray crystallography, I solved the structure of TPR^TSK and characterized a large network of molecular interactions within many binding pockets on TPR^TSK, which stabilize the histone H3.1 tail, including the H3.1 alanine 31 variant-selectivity pocket. Using biochemical assays, I provide an extensive PTM reading profile for TPR^TSK on histone H3.1 showing TPR^TSK is permissive to H3.1 modifications related to active genes and relaxed chromatin but sensitive to those associated with chromatin condensation, including lysine 27 mono-methylation deposited by ATXR5/6 H3.1-specific methyltransferases. Finally, I characterized novel regulatory mechanisms that inhibit ATXR5/6 activity behind the replication fork. This work provides structural and biochemical evidence for understating mechanisms coordinating the DNA damage response during replication that safeguard the genome.