A016 Protein-protein interaction networks identify a role for Huntingtin in regulating chromatin organization and cell identity

<h3>Background</h3> Despite findings gleaned from decades of research, it is still unclear how the polyglutamine-encoding CAG repeat expansion in huntingtin (Htt) causes loss of cell type-specific gene expression, contributing to cellular dysfunction, and ultimately cell death. <h3>Aims</h3> A1) Assess changes to the global proteome in WT, expanded polyglutamine, and Htt knockout hepatocytes. A2) Determine the knockout-validated HTT interactome in nuclear and cytoplasmic fractions from these same hepatocytes. A3) Elucidate roles for HTT in chromatin organization using epigenome profiling approaches. <h3>Methods/Techniques</h3> To shine light on the functions of the HTT protein, as well as how these may become perturbed by polyglutamine expansion, we undertook a global- and affinity-proteomics approach, profiling wildtype and mutant HTT co-immunoprecipitates in Huntington’s disease model mice by mass spectrometry. Using subcellular fractionation and mice with genetic reduction of HTT, we were able to determine a list of high-confidence HTT-interacting proteins, validating these using immunocytochemistry, proximity ligation assay, ChiP-seq, and ATAC-seq. <h3>Results/Outcome</h3> While the expanded polyglutamine mutation causes subtle changes to the hepatocyte proteome, HTT knock-out induces more widespread changes. Surveying HTT interactors in these models suggests roles for HTT in mRNA processing, intracellular trafficking, and chromatin organization. Follow-up on putative nuclear HTT interactors using epigenome profiling shows surprising effects of HTT knockout on chromatin structure focused at cell type-specific genes, suggesting a possible loss of function mechanism arising from polyglutamine expansion. <h3>Conclusions</h3> Overall, our study points to a more direct role for HTT protein in organizing chromatin to support cell type specific gene expression.