Analysis of the Histone H3.1 Interactome: A Suitable Chaperone for the Right Event - PubMed (original) (raw)

Analysis of the Histone H3.1 Interactome: A Suitable Chaperone for the Right Event

Eric I Campos et al. Mol Cell. 2015.

Abstract

Despite minimal disparity at the sequence level, mammalian H3 variants bind to distinct sets of polypeptides. Although histone H3.1 predominates in cycling cells, our knowledge of the soluble complexes that it forms en route to deposition or following eviction from chromatin remains limited. Here, we provide a comprehensive analysis of the H3.1-binding proteome, with emphasis on its interactions with histone chaperones and components of the replication fork. Quantitative mass spectrometry revealed 170 protein interactions, whereas a large-scale biochemical fractionation of H3.1 and associated enzymatic activities uncovered over twenty stable protein complexes in dividing human cells. The sNASP and ASF1 chaperones play pivotal roles in the processing of soluble histones but do not associate with the active CDC45/MCM2-7/GINS (CMG) replicative helicase. We also find TONSL-MMS22L to function as a H3-H4 histone chaperone. It associates with the regulatory MCM5 subunit of the replicative helicase.

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Figures

Figure 1

Figure 1

Quantitative mass spectrometry (MS) analyses of H3.1-interacting proteins. (A) Purification Scheme. (B) Quantitative MS analysis of affinity-purified eH3.1 isolated from asynchronous or synchronized, replicating cells. Each volcano plot represents three independent eH3.1 (FLAG) pull-downs plotted against matching mock purifications. The x axis denotes the eH3.1 over mock ratio of MS intensity whereas a false discovery rate (FDR) adapted t-test is plotted on the y axis. (C) Distribution of H3.1-interacting proteins across subcellular compartments. (D) Relative enrichment of histone chaperones, components of the replication fork, and proteins with enzymatic activity within the eH3.1 immunoprecipitates. Data is presented as mean Label-free Quantification (LFQ) ratio +/− SD.

Figure 2

Figure 2

Biochemical isolation of soluble eH3.1 protein complexes. (A) Anion exchange chromatography of eH3.1 affinity purified from either nuclear extracts (left panel) or solubilized chromatin (right panel). Silver stained SDS-PAGE and western analyses revealed co-eluting proteins (top panels), whereas the extracts were further essayed for intrinsic enzymatic activities towards histones using radiolabeled acetyl-CoA, SAM and ATP (bottom panels). The elution points above the gels denote fractions where eH3.1 protein levels peaked. These fractions, as well as fractions containing enzymatic activities towards histones were further fractionated (see Figure S2, Figure S3). (B) H3.1 interactome based on the biochemical purification of soluble eH3.1 protein complexes. Proteins validated in the quantitative MS analysis are highlighted in yellow. Solid grey and dashed red lines respectively represent interactions reported in STRING, and interactions found through the biochemical purification of soluble eH3.1. NC: negative control, PC: positive control, KAT: lysine acetyltransferase, KMT: lysine methyltransferase, CBB: Coomassie Brilliant Blue.

Figure 3

Figure 3

Nucleosome disassembly. (A) A radiolabeled PCNA probe is loaded onto a linear DNA template, flanked by a single nucleosome at one extremity and a biotin-streptavidin block at the other. DNA flows into the exclusion volume when applied onto a gel filtration spin column, along with the radiolabeled PCNA trapped by the nucleosome. The radioactive probe is free to slide on DNA but falls off and remains in the inclusion volume if histones are removed. The assay quantifies the amount of radioactive PCNA in the exclusion volume by Cherenkov counting. (B) Testing nucleosome disassembly activity within affinity-purified eH3.1 from nuclear fractions (Figure 2). Data represents mean [32P]-PCNA retention +/− SD. (C) Mass spectrometry analysis (peptide counts) of the three fractions exhibiting histone eviction.

Figure 4

Figure 4

Molecular functions of nuclear sNASP and ASF1B. (A) Nuclear sNASP co-elutes with HAT1 and evicted histones. (B) Both sNASP-HAT1 complexes (with or without evicted histones) are enzymatically active. Left panel: Western analysis of peak eH3.1 fractions containing sNASP with evicted or new histones (precipitated at 3.4 and 2.8 M ammonium sulfate, respectively). Right panel: Acetyltransferase activity towards histones demonstrated by autoradiography of acetyl-[3H] incorporation. (C) Nuclear, but not cytosolic sNASP co-precipitates histones with marks characteristic of eu- and hetero-chromatin. (D) In vitro PRC2 methyltransferase assay. sNASP-bound histones are poor substrates for the PRC2 complex compared to free soluble (H3-H4)2 tetramers. (E) In vivo crosslinking of replicating 293 cells (left panel), and mass spectrometry analysis of crosslinked, immunoprecipitated, ASF1B.

Figure 5

Figure 5

TONSL is a histone chaperone that binds H3K9me1. (A) Primary structures of human TONSL and the similar yeast protein, Dia2. (B) Supercoiling assay demonstrating the histone chaperone ability of TONSL. (C) Pull-down assay utilizing immobilized histone peptides testing binding preference by recombinant TONSL and HP1. (D) MS/MS HCD spectrum of the (M + H)+4 ion of cross-linked peptides between TONSL (TRP repeat) and the C-terminus of MCM5. N-terminal fragment ions (b) are indicated in blue and C-terminal fragment ions (y) are indicated in green and red. The mass accuracy for precursor ion is better than 1 ppm and mass accuracy of all the fragment ions is better than 10 ppm. (E) Immobilized TONSL binds to in vitro translated MCM5. (F) Model for TONSL-MMS22L at the replication fork: Upon recruitment to stalled replication forks TONSL-MMS22L may maintain the CMG helicase inactive by binding to MCM5.

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