Mapping the local protein interactome of the NuA3 histone acetyltransferase
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.
SourceAvailable from: Celia Martinez
Dataset: 2014 Vicente-Sara FEBS Open Bio
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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.11/2014; 4. DOI:10.1016/j.fob.2014.11.001
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ABSTRACT: In order to understand how chromatin complexes function in the nucleus, it is important to obtain a comprehensive picture of their protein, DNA, and RNA components, as well as their mutual interactions. This unit presents a chromatin cross-linking approach (BioTAP-XL) that utilizes a special BioTAP-tagged transgenic protein bait along with mass spectrometry to identify protein complex components, and high-throughput sequencing to identify RNA components and DNA binding sites. Full protocols are provided for Drosophila cells and for human cells in culture, along with an additional protocol for Drosophila embryos as the source material. A key element of the approach in all cases is the generation of control data from input chromatin samples. © 2015 by John Wiley & Sons, Inc. Copyright © 2015 John Wiley & Sons, Inc.Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.] 01/2015; 109:21.30.1-21.30.32. DOI:10.1002/0471142727.mb2130s109