Characterization of a Highly Conserved Histone Related Protein, Ydl156w, and Its Functional Associations Using Quantitative Proteomic Analyses

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 largescale 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. Molecular &

The repressor Rgt1 and the cAMP-dependent protein kinases control the expression of the SUC2 gene in Saccharomyces cerevisiae

“…If such were the case, in the absence of PKA Rgt1 would not be able to repress SUC2 transcription and invertase levels would be high. A candidate for a cooperating protein could be histone 3, as it has been reported to interact with Rgt1 [47] and could be a substrate for PKA. However the evidence is still weak, as the interaction was identified in a high throughput experiment and phosphorylation by PKA has not been demonstrated.…”

Mte1 interacts with Mph1 and promotes crossover recombination and telomere maintenance

“…The Mte1-GFP fusion protein has been shown to form nuclear foci in response to replication stress Tkach et al 2012;Yimit et al 2015). Moreover, high-throughput screens have implicated Mte1 in telomere length maintenance (Askree et al 2004) and indicated that it can interact with Cmr1 (Gilmore et al 2012), a chromatin-binding protein associated with replication stress response (Choi et al 2012;Tkach et al 2012;Gallina et al 2015). In this report, we show that Mte1 is a D-loop-binding protein that interacts with the Mph1 helicase and has an antagonizing role to Mph1 in recombination outcomes by enhancing CO formation.…”

Yeast gene CMR1/YDL156W is consistently co-expressed with genes participating in DNA-metabolic processes in a variety of stringent clustering experiments

“…In reference [12], a quantitative proteomics approach was adopted to extend the protein network of core histones (H2A, H2B, H3 and H4) in the budding yeast S. cerevisiae and identified CMR1 as a member in this network. Some 556 proteins were found binding to one or more histones, whereas only 25 proteins of these were found binding to the four core histones.…”

“…The 25 proteins include the four histones (H2A, H2B, H3 and H4), two units of the RPA complex (RFA2 and RFA3), two units of the Ku complex (YKU70 and YKU 80), many units of the RNA polymerase complex (RET1, RPO31, RPC17, RPC37, RPC40 and RPC82), many single-unit proteins (RIM1, YTA7, PSH1, CSE4, ABF2, CKA2, TIF3, DEM1, SUB2 and SMC3) and the previously uncharacterized protein YDL156W/CMR1. Then, associations with the CMR1 protein were investigated, and it was found that many proteins showed stable association with YDL156W, including the six proteins RIM1, RFA2, RFA3, YTA7, YKU70 and YKU80 which are within the 25 proteins found binding to all of the four core histones [12]. …”

“…

Figure 4.Venn diagram illustrating relations between the subsets of genes found by using quantitative proteomics to extend the core histone network and the subset of genes found by tight gene clustering based on gene expression profiles. The subset ( a ) represents the 25 genes found to be associated with the four core histones [12], the subset ( b ) represents the seven genes out of those 25 found to be associating with CMR1 [12] and the subset ( c ) represents the 19 co-expressed genes found in the tightest cluster of genes by using the Bi-CoPaM method in our study.

…”