10/31/2024
By Kwok Fan Chow

The Kennedy College of Science, Department of Chemistry, invites you to attend a Ph.D. Dissertation defense by Matthew R Marunde entitled “Decoding Chromatin Interactions: Harnessing Nucleosome Substrates and High-Throughput Approaches.”

Degree: Doctoral
Location: Virtual, Zoom link
Date: Friday, November 8, 2024
Time: 10 a.m.

Committee Chair:
in Xu, Ph.D., Department of Chemistry, University of Massachusetts Lowell

Committee Members:
Matthew Gage, Ph.D., Department of Chemistry, University of Massachusetts Lowell
Juan Artes Vivancos, Ph.D., Department of Chemistry, University of Massachusetts Lowell
Carl Lawton, Ph.D., Department of Chemical Engineering, University of Massachusetts Lowell

Abstract:
The fundamental subunit of chromatin is the nucleosome, composed of a histone octamer wrapped by ~147bp of DNA. Nucleosomes play an essential role in organizing and compacting the genome; however, they are far more than a simple DNA packaging unit, they are critical regulators of many cellular processes. These processes are mediated by chromatin-associated proteins (CAPs) that are localized to specific genomic regions via their reader domains, which recognize and bind to histone post-translational modifications (PTMs). Specific patterns of PTMs form a dynamic language known as the histone code. Dysregulation of this code has been implicated in the onset and progression of diseases such as cancer. Considerable effort has been devoted to deciphering the histone code and characterizing reader domains. Traditional methods have relied on histone peptides rather than nucleosomes, due to the significant reagent requirements and cost, and complexity of nucleosome production. However, histone peptides fail to replicate the complex structure of nucleosomes and chromatin. Peptides lack important features such as histone-DNA interactions, key regions like the H2A/H2B acidic patch, and physiological charge, potentially leading to misleading results. In this dissertation, I first present our work on the development and application of the dCypher approach, which leverages robust high-throughput technologies (i.e. AlphaScreen and Luminex). Due to its high sensitivity and adaptability, this approach this approach is compatible with both histone peptides and nucleosomes, dramatically reducing reader and target consumption up to 1000-fold compared to traditional approaches (e.g. peptide arrays), while enhancing signal-to-background ratios. Secondly, I describe our characterization of acyl and methyl reader proteins using both histone peptides and nucleosomes. Our results demonstrate the importance of nucleosome context for accurately characterizing reader domains, which frequently refined binding compared to peptides. Finally, I present our work with the tandem reader domains (PHD-BD) of BPTF, a subunit of the nucleosome remodeling factor (NURF) complex. Dysregulation of this complex has been associated with several forms of cancers, and therapeutic targeting of its binding domains has shown promise. This work further highlights the importance of using nucleosomes,which were necessary to identify the synergy between the PHD-BD domains. Additionally, using homotypic and heterotypic nucleosomes, we observed that PTMs must be present on the same tail for PHD-BD to bind synergistically, revealing an additional level of chromatin regulation. Through genomic mapping (i.e. CUT&RUN), we confirmed our in vitro findings by demonstrating that reader domains localize to regions similar to nucleosome validated PTM antibodies. Overall, this dissertation introduces a novel platform for the characterization of reader domains, demonstrates the importance of nucleosomes for understanding chromatin interactions in vitro, and demonstrates the potential of using individual or multivalent reader domains as alternative affinity reagents for genomic mapping.

All interested students and faculty members are invited to attend.