Histones package genomic DNA in the eukaryotic nucleus into chromatin, whose structure controls all DNA- based processes, including transcription, replication, recombination, and repair. A major mechanism regulating chromatin structure is based on post-translational modifications of the histones, which are deposited by enzymes or “writers” and recognized with high specificity and selectivity by “reader” domains of certain proteins to mediate downstream functions. The interplay between writers and readers is essential for development and is perturbed in many diseases, including cancer. A major focus in chromatin biology is thus to understand the detailed mechanisms that control chromatin structure, which is greatly hampered by the relatively few high-resolution structures of chromatin-bound proteins and protein complexes that deposit and recognize histone modifications. Large segments of the eukaryotic genome are packaged into transcriptionally silent heterochromatin. Heterochromatin is critical to maintain genome integrity and to prevent chromosomal defects. Interestingly, heterochromatin formation and DNA replication are often coordinated through specific chromatin modifications. For example, different methylation states of histone H4 at lysine 20 (H4K20) regulate distinct processes: H4K20me3 is found in heterochromatin, whereas H4K20me2 is found at the origins of replication. Methylation of H4K20 is catalyzed by two related histone methyltransferases, SUV4-20H1 and SUV4-20H2, but it is not known how these enzymes are recruited to chromatin or how their catalytic activity is regulated. In addition, heterochromatin formation and replication can be coordinated through readers, such as Orc1, a subunit of the Origin Recognition Complex (ORC). Orc1 binds to chromatin using its bromo-adjacent homology (BAH) domain, which interacts with histone modifications and heterochromatin proteins. The detailed mechanisms involved in these processes are largely unknown. We will use structural and functional approaches to fill this critical knowledge gap, with AIM 1 focusing on elucidating the mechanisms of SUV4-20H enzymes and AIM 2 focusing on understanding the mechanisms of Orc1 BAH domains. We will use cryo-EM to determine structures of these writers and readers bound to nucleosomes and complement the structures with functional experiments in vitro and in vivo. The structural and functional studies of SUV4-20H writers and Orc1 BAH domain readers in complex with nucleosomes will uncover general principles that underlie the deposition and recognition of histone modifications. Our proposed comprehensive studies will provide crucial insights into the fundamental biological processes of heterochromatin formation and replication and will provide invaluable insights into how deregulation of these complexes and resulting aberrations in chromatin structure contribute to diseases.

Heterochromatin structure is critical to gene silencing, genome integrity and to prevent chromosomal defects that can result in diseases. In this application, we propose to use structural and functional approaches to elucidate the intricate mechanisms of heterochromatin regulation. These novel mechanistic insights will open new avenues towards therapies based on the regulation of protein complexes that play critical roles in heterochromatin.