Abstract
Heterochromatin protein 1 (HP1) family members (alpha, beta, and gamma) bind histone H3 methylated at Lys-9, leading to gene silencing and heterochromatin formation. Several previous reports have suggested that HP1s are post-translationally modified, yet sites of modification have not yet been exhaustively determined. Here we perform the first comprehensive proteomic analysis of all HP1 isoforms using tandem mass spectrometry. Our data reveal that all HP1 isoforms are highly modified in a manner analogous to histones including phosphorylation, acetylation, methylation, and formylation, including several sites having multiple different types of modifications. Additionally, many of these modifications are found in both the chromo- and chromoshadow domains, suggesting that they may have an important role in modulating HP1 interactions or functions. These studies are the first to systematically map the abundant sites of covalent modifications on HP1 isoforms and provide the foundation for future investigations to test whether these modifications are essential in heterochromatin maintenance or other nuclear processes.
Highlights
Heterochromatin protein 1 (HP1) family members (␣, , and ␥) bind histone H3 methylated at Lys-9, leading to gene silencing and heterochromatin formation
The mammalian HP1␣, , and ␥ proteins are a family of chromatin-associated proteins that are homologs of the Drosophila heterochromatin protein 1 (HP1)1, originally identified as a protein required for position effect variegation
The HP1 proteins have been shown to interact with a number of other proteins/complexes that are involved with chromatin metabolism such as; the CAF-1 complex, a histone deposition chaperone; the origin recognition complex, a protein complex required for DNA replication that binds to origins of replication; and the BRG1 complex, an ATP-dependent chromatin remodeling machine [12,13,14]
Summary
Cell Culture and Protein Isolation—HP1␣, HP1, and HP1␥ coding sequences were amplified by PCR from a human cDNA library. For in-solution digestion, HP1 was diluted with 50 l of 100 mM ammonium bicarbonate and reduced with 10 mM dithiothreitol for 1 h at 51 °C followed by alkylation with iodoacetamide (25 mM) for 45 min at room temperature in the dark. At this point, HP1 was digested for 6 –12 h at 37 °C using either trypsin at a protein:enzyme ratio of 20:1, LysC at a protein:enzyme ratio of 10:1, or chymotrypsin at a protein: enzyme ratio of 20:1. HP1 was separated on a 15% SDS-polyacrylamide gel, and in gel digested after dithiothreitol and iodoacetamide treatment (same concentrations as above) with tryp-
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