UCSF

DNA encodes the genetic material to instruct all cellular processes and to establish cellular identity. Cellular identity is established by both genetic content and regulation of gene expression. In eukaryotes, gene expression is regulated by many factors including chromatin structure. Chromatin structure consists of nucleosomes, comprised of ~150 bp of DNA wrapped around a histone octamer. This structure regulates several DNA dependent processes including transcription and DNA damage repair. Understanding the mechanisms that regulate chromatin structure is key to understanding how biological systems are controlled in the cell.

DNA dependent cellular process are known to be regulated by chromatin remodelers. Chromatin remodelers couple the energy of ATP hydrolysis to slide nucleosomes, transfer histones, and/or distort the octamer— activities that are essential to regulate the chromatin state. A unique chromatin remodeler, INO80, requires both the RecA-like ATPase and accessory subunits to slide nucleosomes in vitro. In vivo, INO80 plays a role in both DNA damage repair and transcription. However, it is unclear how INO80 sliding activity contributes to its diverse biological roles. Recent work from our lab revealed the following about INO80 mechanism: (1) INO80 is regulated by two nucleosome cues: flanking DNA length and the H2A-2B dimer acidic patch on the histone octamer; (2) INO80 remodeling reaction has at least one intermediate state and transition of this intermediate to a slid product is regulated by DNA length; (3) the Nhp10 accessory module regulates the transition between the intermediate state and the slid product. Others have shown that both Nhp10 and Arp8 modules recognize DNA. The Arp8 module alters how INO80 engages both the DNA and histones of the nucleosome, specifically altering how the Arp5 module engages the H2A-H2B dimer. Cryo-EM structures revealed that the Arp5 and Ies2 modules engage the H2A-H2B dimer acidic patch. It wasn’t clear if the engagement of both Arp5 and Ies2 with the two respective H2A-H2B dimer acidic patches was required for INO80 remodeling. In this thesis, we investigated the requirement of the Arp5-H2A-H2B-acidic patch interaction versus the Ies2-H2A-H2B-acidic patch interaction in INO80 remodeling. We discovered that only the Arp5-H2A-H2B acidic patch interaction was required for INO80 remodeling and contributed to the generation of a nucleosome intermediate. Others have also shown that INO80 preferentially acts on nucleosome containing H2A.Z-H2B dimer, which has a more expansive acidic patch. H2A.Z-H2B containing nucleosomes are present during transcription. As INO80 acts during transcription, what alternative substrates generated during transcription could INO80 act on? Others have shown that hexasomes, nucleosomes missing one H2A-H2B dimer, are generated by polymerase during transcription. In this thesis, we further investigated the role INO80 plays in remodeling hexasomes. We identified that INO80 not only regulates hexasome positions in yeast, but INO80 is also preferentially stimulated by hexasomes. Surprisingly, the dimer missing in the hexasome is the dimer Arp5 contacts in the nucleosome. This led us to hypothesize that Arp5 must make alternative contacts on the hexasome, that allow INO80 to be more active. In this thesis, we additionally obtained the structure of INO80 bound to a hexasome. We showed that not only does Arp5 make alternative contacts with the H3-H4 tetramer, but the Ino80 ATPase engages the hexasome at SHL-2, which reflects a ~180o rotation compared to the nucleosome. Most remodelers bind at SHL2 of the nucleosome. This provides an explanation for why INO80 is more active on a hexasome compared to a nucleosome. We hypothesize that INO80 generates a hexasome-like intermediate while remodeling a nucleosome. We speculate that Ino80 migrates from SHL-7/-6 towards SHL-2 on path to remodeling a nucleosome. Overall this thesis highlights the versatility with which remodelers like INO80 act on various substrates. Such versatility which may allow INO80 to act in diverse processes in vivo.