The Role of Post-Translational Modifications in Regulating Distinct Functions of Heterochromatin Protein One

Abstract

Heterochromatin is a minimally transcribed, densely bundled complex of DNA and associated factors comprising large regions of the eukaryotic genome. It is essential for chromosome stability, genome integrity, gene regulation, and the silencing of transposons. The filamentous fungus Neurospora crassa is often employed as a model organism to study the epigenetic regulation ofheterochromatin. In Neurospora, the conserved scaffolding protein heterochromatin protein 1 (HP 1) binds H3 histones marked by lysine nine trimethylation (H3K9me3) and recruits other proteins to form at least four distinct complexes. HPl recruits the DIM-2 DNA methyltransferase, which catalyzes DNA methylation, and the Mi-2 chromatin remodeler, which promotes centromeric silencing. HPI is also an essential component of the HCHC complex, which facilitates histone deacetylation, and the DMM complex, which limits aberrant heterochromatin spreading. However, it is unclear how these disparate functions are coordinated. We hypothesized that they are modulated by post-translational modifications (PTMs) of HPI. Previously, we used mass spectrometry to identify HPl sites harboring methylation, acetylation, formylation, and phosphorylation. I used amino acid substitutions at a subset of these sites to prevent individual PTMs in vivo. Substitutions at multiple sites were found to cause a substantial decrease in centromeric silencing independent of DNA methylation. These results suggest that the recruitment of Mi-2 to incipient heterochromatin may be selectively mediated by specific PTMs.