NSF-BSF: Dissecting the HIRA/Hir chaperone complex in the chromatin landscape

This project aims to uncover the fundamental molecular mechanisms that control how genetic information is accessed, packaged, and protected inside living cells. In all eukaryotic organisms, genomic DNA is tightly wrapped around histone proteins to form structures called nucleosomes. While these structures package DNA, they also play a critical regulatory role in controlling gene activity. This research focuses on the conserved HIRA/Hir complex, a vital cellular machine responsible for assembling nucleosomes independently of DNA replication at active genes. Beyond its essential role in managing normal gene expression, this complex plays a critical role in genome defense by depositing histones onto viral DNA to restrict viral genomes. Despite these vital functions, the precise mechanisms regulating its activity remain poorly understood. By revealing how these machines function using yeast as a model system, this project provides fundamental knowledge essential for understanding cellular health, aging, and antiviral defense. Beyond the laboratory, this project supports education, international collaboration, and workforce development by providing structured, cross-institutional training for students in cutting-edge biochemistry and computational structural biology. The project leverages an established ten-year international partnership, facilitating exchange visits and integrating advanced technology into university courses to develop the next generation of interdisciplinary scientists capable of tackling complex challenges in biotechnology and biomedicine. This research project investigates the structural and biochemical mechanisms of the HIRA/Hir chaperone complex within the chromatin landscape. The primary objective is to establish a comprehensive, mechanistic understanding of HIRA/Hir-mediated replication-independent histone H3/H4 deposition at promoters and transcriptionally active regions. To address the challenges of studying highly dynamic, transient, and flexible chromatin-transcription interactions, this project integrates single-particle cryo-electron microscopy (cryo-EM) with a novel cross-linking/mass spectrometry (CL-MS) pipeline, alongside genetic and biochemical analyses. The application of improved CL-MS methods serves a dual purpose: in situ CL-MS will map protein-protein interactions within native chromatin, while in vitro CL-MS will guide high-confidence modeling of flexible domains and intrinsically disordered regions, assisting in cryo-EM structure determination. This coordinated approach will elucidate the sequential steps of nucleosome assembly and define its physical coordination with the transcription machinery. This work directly advances the field of biotechnology by establishing a predictive, high-resolution framework for modulating chromatin dynamics and epigenetic inheritance, while demonstrating a broadly applicable paradigm for integrating complementary structural approaches to dissect other macromolecular systems. This collaborative US/Israel project is supported by the US National Science Foundation and the Israeli Binational Science Foundation. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria. NSF Award ID: 2611572 | Program: 01002627DB NSF RESEARCH & RELATED ACTIVIT | Principal Investigator: KENJI MURAKAMI | Institution: University of Pennsylvania, PHILADELPHIA, PA | Award Amount: $1,300,000 View on NSF Award Search: https://www.nsf.gov/awardsearch/show-award/?AWD_ID=2611572 View on Research.gov: https://www.research.gov/awardapi-service/v1/awards/2611572.html