
Melesse Nune
The eukaryotic DNA is beautifully organized into a nucleosome comprising 147 bp DNA wrapped around an octamer of core histone proteins H2A, H2B, H3 and H4. The nucleosome is the basic repeating unit of a chromatin. The chromatin is decorated with a wide range of reversible histone post-translational modifications that regulate all processes that require access to DNA. Monoubiquitination of histone H2B (H2B-Ub) plays a role in transcription and DNA replication, and is required for the function of a protein complex that is responsible for the assembly and disassembly of nucleosomes called FACT. Dysregulation of H2B-Ub or FACT is associated with a variety of cancers. In Cynthia Wolberger’s lab, my study focused on understanding how FACT and the deubiquitinating enzyme Ubp10 work in concert to govern histone H2B deubiquitination. My work demonstrated that Ubp10 preferentially cleaves free-standing H2A/H2B-Ub dimers much faster than intact ubiquitinated nucleosomes, but that the addition of FACT stimulates Ubp10 activity on nucleosomes. Importantly, my work also demonstrated that disrupting the functions of these proteins in cells leads to defects in transcription and DNA replication. To get a better understanding of the reason behind Ubp10’s low activity on the nucleosomes, I solved the cryogenic electron microscopy (cryoEM) structures of Ubp10 bound to a ubiquitinated nucleosome. The structures revealed that Ubp10 makes several contacts with histones, ubiquitin, and severely alters the nucleosomal DNA at the nucleosome entry/exit site. Ubp10 docks onto the nucleosome in many conformations suggesting that the enzyme doesn’t bind nucleosomes in the correct register that promotes H2B deubiquitination without the help of FACT. The findings from my work highlight novel relationships between H2B monoubiquitination and the role of FACT in destabilizing the nucleosome to assist Ubp10 in H2B deubiquitination.