Article

Mapping the local protein interactome of the NuA3 histone acetyltransferase

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
Protein Science (Impact Factor: 2.85). 09/2009; 18(9):1987-97. DOI: 10.1002/pro.212
Source: PubMed

ABSTRACT

Protein-protein interactions modulate cellular functions ranging from the activity of enzymes to signal transduction cascades. A technology termed transient isotopic differentiation of interactions as random or targeted (transient I-DIRT) is described for the identification of stable and transient protein-protein interactions in vivo. The procedure combines mild in vivo chemical cross-linking and non-stringent affinity purification to isolate low abundance chromatin-associated protein complexes. Using isotopic labeling and mass spectrometric readout, purified proteins are categorized with respect to the protein 'bait' as stable, transient, or contaminant. Here we characterize the local interactome of the chromatin-associated NuA3 histone lysine-acetyltransferase protein complex. We describe transient associations with the yFACT nucleosome assembly complex, RSC chromatin remodeling complex and a nucleosome assembly protein. These novel, physical associations with yFACT, RSC, and Nap1 provide insight into the mechanism of NuA3-associated transcription and chromatin regulation.

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    • "28 (0.32) binds methylated histone), Elp3p (the HAT subunit of elongator complex) and Spt5p (RNAPII core binding, transcription elongation by RNAPII). In particular, the identification of Spt16p and Pob3p, protein components of the yFACT nucleosome assembly complex, and Rfc1p, according to others studies [39], provides insights into the mechanism of NuA3-associated transcription and chromatin regulation. Previous studies have reported that Sas3p, core histones, Spt16p and Pdp3p, are co-purified with Yta7p tagged with Protein A [40]. "

    Full-text · Dataset · May 2015
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    • "28 (0.32) binds methylated histone), Elp3p (the HAT subunit of elongator complex) and Spt5p (RNAPII core binding, transcription elongation by RNAPII). In particular, the identification of Spt16p and Pob3p, protein components of the yFACT nucleosome assembly complex, and Rfc1p, according to others studies [39], provides insights into the mechanism of NuA3-associated transcription and chromatin regulation. Previous studies have reported that Sas3p, core histones, Spt16p and Pdp3p, are co-purified with Yta7p tagged with Protein A [40]. "
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    ABSTRACT: Histone acetylation affects several aspects of gene regulation, from chromatin remodelling to gene expression, by modulating the interplay between chromatin and key transcriptional regulators. The exact molecular mechanism underlying acetylation patterns and crosstalk with other epigenetic modifications requires further investigation. In budding yeast, these epigenetic markers are produced partly by histone acetyltransferase enzymes, which act as multi-protein complexes. The Sas3-dependent NuA3 complex has received less attention than other histone acetyltransferases (HAT), such as Gcn5-dependent complexes. Here, we report our analysis of Sas3p-interacting proteins using tandem affinity purification (TAP), coupled with mass spectrometry. This analysis revealed Pdp3p, a recently described component of NuA3, to be one of the most abundant Sas3p-interacting proteins. The PDP3 gene, was TAP-tagged and protein complex purification confirmed that Pdp3p co-purified with the NuA3 protein complex, histones, and several transcription-related and chromatin remodelling proteins. Our results also revealed that the protein complexes associated with Sas3p presented HAT activity even in the absence of Gcn5p and vice versa.We also provide evidence that Sas3p cannot substitute Gcn5p in acetylation of lysine 9 in histone H3 in vivo. Genome-wide occupancy of Sas3p using ChIP-on-chip tiled microarrays showed that Sas3p was located preferentially within the 50-half of the coding regions of target genes, indicating its probable involvement in the transcriptional elongation process. Hence, this work further characterises the function and regulation of the NuA3 complex by identifying novel post-translational modifications in Pdp3p, additional Pdp3p-co-purifying chromatin regulatory proteins involved in chromatin-modifying complex dynamics and gene regulation, and a subset of genes whose transcriptional elongation is controlled by this complex.
    Full-text · Article · Nov 2014 · FEBS Open Bio
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    • "This technique primarily differs from traditional affinity capture-MS method in growing the cells in isotopic media and in analyzing the MS results. A recent report on the identification of the NuA3 acetyltransferase interactome using the I-DIRT technique showed that 278 proteins of 288 proteins identified were actually nonspecific interactors (63). We employed this technique to determine the specific interacting partners for Pif1. "
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    ABSTRACT: Pif1 helicase plays various roles in the maintenance of nuclear and mitochondrial genome integrity in most eukaryotes. Here, we used a proteomics approach called isotopic differentiation of interactions as random or targeted to identify specific protein complexes of Saccharomyces cerevisiae Pif1. We identified a stable association between Pif1 and a mitochondrial SSB, Rim1. In vitro co-precipitation experiments using recombinant proteins indicated a direct interaction between Pif1 and Rim1. Fluorescently labeled Rim1 was titrated with Pif1 resulting in an increase in anisotropy and a Kd value of 0.69 µM. Deletion mutagenesis revealed that the OB-fold domain and the C-terminal tail of Rim1 are both involved in interaction with Pif1. However, a Rim1 C-terminal truncation (Rim1ΔC18) exhibited a nearly 4-fold higher Kd value. Rim1 stimulated Pif1 DNA helicase activity by 4- to 5-fold, whereas Rim1ΔC18 stimulated Pif1 by 2-fold. Hence, two regions of Rim1, the OB-fold domain and the C-terminal domain, interact with Pif1. One of these interactions occurs through the N-terminal domain of Pif1 because a deletion mutant of Pif1 (Pif1ΔN) retained interaction with Rim1 but did not exhibit stimulation of helicase activity. In light of our in vivo and in vitro data, and previous work, it is likely that the Rim1–Pif1 interaction plays a role in coordination of their functions in mtDNA metabolism.
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