Abstract
A significant challenge in biology is to functionally annotate novel and uncharacterized proteins. Several approaches are available for deducing the function of proteins in silico based upon sequence homology and physical or genetic interaction, yet this approach is limited to proteins with well-characterized domains, paralogs and/or orthologs in other species, as well as on the availability of suitable large-scale data sets. Here, we present a quantitative proteomics approach extending the protein network of core histones H2A, H2B, H3, and H4 in Saccharomyces cerevisiae, among which a novel associated protein, the previously uncharacterized Ydl156w, was identified. In order to predict the role of Ydl156w, we designed and applied integrative bioinformatics, quantitative proteomics and biochemistry approaches aiming to infer its function. Reciprocal analysis of Ydl156w protein interactions demonstrated a strong association with all four histones and also to proteins strongly associated with histones including Rim1, Rfa2 and 3, Yku70, and Yku80. Through a subsequent combination of the focused quantitative proteomics experiments with available large-scale genetic interaction data and Gene Ontology functional associations, we provided sufficient evidence to associate Ydl156w with multiple processes including chromatin remodeling, transcription and DNA repair/replication. To gain deeper insights into the role of Ydl156w in histone biology we investigated the effect of the genetic deletion of ydl156w on H4 associated proteins, which lead to a dramatic decrease in the association of H4 with RNA polymerase III proteins. The implication of a role for Ydl156w in RNA Polymerase III mediated transcription was consequently verified by RNA-Seq experiments. Finally, using these approaches we generated a refined network of Ydl156w-associated proteins.
Highlights
From the ‡Stowers Institute for Medical Research, Kansas City, MO 64110; §Department of Pathology and Laboratory Medicine, The University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160, USA
Through focused quantitative proteomics experiments combined with a bioinformatics approach utilizing genetic interaction data, gene deletions, and functional associations provided through the Gene Ontology consortium, we propose that the histone-associated protein Ydl156w is necessary for proper chromatin remodeling, transcription through RNA polymerase III, and DNA replication and repair
Our data shows that the four histones, i.e. H2A, H2B, H3, and H4, strongly associate with additional 15 proteins, which were found in affinity purifications of all four histones and were among the 31 proteins enriched in the entire data set as determined by Singular Value Decomposition (SVD)
Summary
Through focused quantitative proteomics experiments combined with a bioinformatics approach utilizing genetic interaction data, gene deletions, and functional associations provided through the Gene Ontology consortium, we propose that the histone-associated protein Ydl156w is necessary for proper chromatin remodeling, transcription through RNA polymerase III, and DNA replication and repair.
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