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

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Background 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. Aims 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. Methods/Techniques 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. Results/Outcome 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. Conclusions Overall, our study points to a more direct role for HTT protein in organizing chromatin to support cell type specific gene expression.

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