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.

Figure 1

Transient I-DIRT is a method for unambiguous identification of stable and transient protein–protein interactions. One culture of isotopically light cells containing an affinity-tagged protein is subjected to in vivo chemical cross-linking with formaldehyde (FA), as is a second culture of isotopically heavy cells that does not contain the tagged protein. These cells are frozen independently, subsequently mixed in a ratio of 1:1 and co-lysed under cryogenic conditions. During the affinity purification, proteins that exist in stable interactions with the tagged protein are isotopically light, while contaminants are a 1:1 mixture of light and heavy. Proteins having a transient interaction with the tagged protein complex show an intermediate level of isotopically light peptides.

Figure 2

Mild formaldehyde cross-linking is optimal for the liberation and affinity purification of chromatin-associated protein complexes. YNG1-TAP cells were subjected to various amounts of in vivo formaldehyde (FA) cross-linking. A: Cellular lysates were sonicated to shear DNA. Purified DNA was resolved by agarose gel electrophoresis and visualized with ethidium bromide. B: Sonicated cell lysates were clarified by centrifugation and TAP tagged Yng1 was affinity purified (AP) on IgG-coated Dynabeads. Aliquots were collected during the indicated steps of the purification and the relative amount of Yng1-TAP was visualized by western blotting.

Figure 3

In vivo chemical cross-linking of NuA3 traps novel transiently interacting proteins. YNG1-TAP cells (±0.05% formaldehyde) were collected for affinity purification. A: Cell lysates were subjected to sonication to shear DNA. Isolated DNA was resolved by agarose gel electrophoresis and visualized with ethidium bromide. B: Yng1-TAP and associated proteins were purified on IgG-coated Dynabeads, resolved by SDS-PAGE, visualized by Coomassie staining and excised for protein identification. Proteins were digested with trypsin for peptide analysis by LC-MS² (Thermo LTQ-XL). Proteins identified (99% confidence) were categorized into a Venn diagram. The core NuA3 components (Sas3, Nto1, Yng1, Taf30, Eaf6) were identified in both purifications, while yFACT/Nap1/Rsc were trapped with chemical cross-linking. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 4

Transient I-DIRT technology differentiates specific, transient and nonspecific protein–protein interactions with the NuA3 histone K-acetyltransferase. A: An arginine auxotrophic strain showed complete incorporation of ¹³C₆-arginine. Proteins from an arg4Δ strain grown either in isotopically light or heavy synthetic yeast medium were resolved by SDS-PAGE. Identical bands containing pyruvate decarboxylase PDC1 were excised and treated with trypsin. Peptides were analyzed by MALDI mass spectrometry. Shown is a representative peptide that demonstrates the complete incorporation of heavy arginine. B: Purification of in vivo cross-linked Yng1-TAP from a 1:1 mixture of YNG1-TAP isotopically light and arg4Δ isotopically heavy cells. Proteins co-purifying with Yng1-TAP were resolved by SDS-PAGE, visualized by Coomassie staining and excised for protein identification. Gel bands were treated with trypsin and peptides were analyzed with a Thermo LTQ Orbitrap mass spectrometer. C: Mass spectra of peptides from proteins that are specific, transient or nonspecific interactors with the Yng1-TAP containing protein complex.

Figure 5

NuA3 transiently interacts with the yFACT and RSC complexes in vivo. A: Arginine-containing peptides were identified for 288 of the proteins identified from the gel shown in Figure 4(B). The fraction of isotopically light peptide relative to light + heavy is plotted for each. B: Condensed version of panel A. Core NuA3 components (Sas3, Nto1, Yng1, Taf14, Eaf6) were identified as ∼100% light. A nonspecific threshold (0.47 ± 0.08) was established with a group of ribosomal proteins, which are common contaminants in affinity purifications. Relative to the nonspecific threshold, the majority (278) of the proteins were classified as nonspecific. Five proteins showed a fraction of isotopically light that were >2 standard deviations from the contaminant threshold and were categorized as transient interactions: Pob3/Spt16 (yFACT components), Nap1 (nucleosome assembly protein 1) and Rsc7/8 (RSC chromatin remodeling complex components).

Figure 6

NuA3 and yFACT have overlapping in vivo functions. A: Indicated strains were 10-fold serially diluted and assayed for temperature, hydroxyurea (HU) or methyl methanesulfonate (MMS) sensitivity. B: Proposed model of NuA3 in transcriptional activation/elongation. Set1-modified H3K4me3 recruits NuA3 for acetylation of H3K14. RSC is localized to the H3K14Ac and serves as a bridge to RNA polymerase II. yFACT is localized via protein-protein interactions. Nap1 shuttles in histones to this macromolecular protein assembly. The chromatin remodeling activity of RSC and nucleosome disassembly activity of yFACT destabilize a nucleosome to provide for RNA polymerase II movement. yFACT replaces the nucleosome with histones delivered by Nap1.