Structural and Biochemical Characterization of Histone Acetyltransferase Complexes on the Nucleosome

Restricted (Penn State Only)
- Author:
- Espinola Lopez, Jose
- Graduate Program:
- Molecular, Cellular, and Integrative Biosciences
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 06, 2023
- Committee Members:
- Andrey Krasilnikov, Major Field Member
Song Tan, Chair & Dissertation Advisor
Susan Hafenstein, Major Field Member
Tae-Hee Lee, Outside Unit & Field Member
Melissa Rolls, Program Head/Chair - Keywords:
- Nucleosome
Chromatin
cryoEM
Nanobody
Acetyltransferase
HAT - Abstract:
- Histone acetyltransferases (HATs) are essential enzymes that regulate chromatin structure and gene expression by catalyzing the acetylation of histone proteins. Recent studies have provided new insights into how HATs interact with and acetylate nucleosomes, the fundamental units of chromatin. To investigate HAT mechanisms, I developed a fluorescence-based assay to enable the continuous monitoring of HAT activity on nucleosomes, eliminating the need for radioactive materials while minimizing sample requirements. Combined with TR-FRET binding experiments, qualitative binding assays, and cryoEM my studies open new hypotheses to better understand the mechanisms by which HATs sense and respond to features of the nucleosome to optimize binding and catalysis. The Gcn5/Ada2/Ada3 HAT subcomplex within the SAGA coactivator prefers nucleosomes with longer flanking DNA, exhibiting enhanced binding and activity. Low-resolution structural analysis indicates multivalent binding modes involving linker DNA, histone H3, and H4 interactions. Additionally, the acidic patch does not appear to interact with the HAT complex. This was further confirmed by binding and activity assays. Deletion of Ada2’s SWIRM domain abrogated flanking DNA length dependence, implicating it in linker sensing. Similarly, the MOF/MSL1v1 HAT complex binding was augmented by longer flanking DNA, but flanking DNA had only modest impacts on catalysis. This implies that while flanking DNA aids recruitment, elements within the nucleosome core like the acidic patch primarily govern acetyltransferase activity. Low-resolution structural analysis indicates that MOF/MSL1v1 does, in fact, interact with the acidic patch. While mutation of H2A residues in the acidic patch did not affect MOF binding affinity, it reduced catalytic efficiency, implying the acidic patch is important for optimal catalysis by MOF. Together, these findings suggest MOF utilizes distinct binding modes dependent on flanking DNA interactions to recognize nucleosomal substrates, and elements within the core nucleosome to provide essential features needed for acetyltransferase activity. Finally, to improve sample stability in cryoEM studies I used a yeast surface display platform to generate nanobodies that strongly bind the Ada2/Ada3/Gcn5 HAT complex. However, these nanobodies inhibited HAT/nucleosome complex formation, likely by blocking key nucleosome interaction sites. Future efforts to fine-tune nanobody binding selection, screening, and properties enable their use to aid in obtaining high-resolution HAT/nucleosome complex cryoEM structures. The advances presented here provided new preliminary insights into how HATs engage with the nucleosome. A HAT assay development enabled detailed kinetic analysis on physiologically relevant nucleosomal substrates. Structural and biochemical analyses suggest multivalent recruitment mechanisms responsive to flanking DNA, while also highlighting key nucleosome elements that specify binding and catalytic activity. Moving forward, nanobodies, improved structural techniques, and additional activity and binding assays promise to elucidate additional molecular details of HAT binding and function on the nucleosome.