Many key cellular processes are regulated by protein-protein interactions. The ability of one protein to recognize another protein can trigger cascades of events as seen for signal transduction or can simply target a protein for a process such as degradation. These protein-protein associations can be modulated on many levels including protein abundance, subcellular location, and post-translational modification. Also of critical importance is the affinity of the association. Defining these associations is crucial for understanding any cellular process involving protein interactions. Technology termed transient I-DIRT (transient isotopic differentiation of interactions as random or targeted) has been developed to quantitatively identify transient protein-protein interactions on chromosomes. This technology combines mild in vivo chemical cross-linking, affinity purification of chromosome associated protein complexes, and isotopic labeling with mass spectrometric readout to classify co-purifying proteins as stable, transient, or contaminant interactors. This technology has been optimized for researchers studying low abundance, chromatin associated protein complexes using standard affinity purification protocols. The transient I-DIRT technique is one of the first technologies for identifying and quantifying transient protein-protein interactions in vivo and in the context of a chromosome.
The transient I-DIRT technology applied to the Saccharomyces cerevisiae NuA3 protein complex revealed that this histone acetyltransferase transiently interacts with the nucleosomal assembly complex yFACT, the RSC chromatin remodeling complex, and the nucleosomal assembly protein Nap1. Further investigation revealed that the interaction between NuA3 and yFACT occurs at the nucleosomal level and is not bridged by internucleosomal DNA. NuA3 and yFACT were found to interact in a cell cycle independent manner. A series of yeast epistasis assays illustrated that NuA3 and yFACT protein complexes have overlapping in vivo function. The C-terminal acidic domain of Sas3 was found to be necessary for the interaction between NuA3 and yFACT but not for NuA3 complex stability. Genome wide ChIP-chip analysis demonstrated that the plant homeodomain (PHD) binding domain of Yng1 is necessary for proper genomic distribution of NuA3. These novel physical and functional interactions provide insight into the mechanism of NuA3 associated transcriptional and chromatin regulation.
|School:||University of Arkansas for Medical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 71/10, Dissertation Abstracts International|
|Keywords:||Histone acetyltransferase, Protein-protein interactions|
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