The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Summary

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

Abstract

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

Introduction

Chromatin is a highly dynamic nucleoprotein complex that is involved as primary template for all DNA-mediated processes. The nucleosome is the basic repeated unit of chromatin and consists of a proteinaceous octameric core containing two molecules of each canonical histone H2A, H2B, H3 and H4, around which 147 bp of DNA are wrapped^1,2. All core histones are structured as a globular domain and a flexible N-terminal “tail” that protrudes outside the nucleosome. One of the major mechanisms for regulating chromatin structure and dynamics is based on covalent post-translational modifications (PTMs), which mainly occur on the N-termini of histones^3,4. Histone modifications can function either by altering the higher order chromatin structure, by changing contacts between histones-DNA or between nucleosomes, and thus controlling the accessibility of DNA-binding proteins (cis mechanisms), or by acting as docking sites for regulatory proteins, either as single units, or embedded in multimeric complexes. Such regulating proteins can exert their function in different ways: by modulating directly gene expression (i.e. TAF proteins), or by altering the nucleosome positioning (i.e. chromatin remodeling complexes) or by modifying other histone residues (i.e. proteins with methyl-transferase or acetyl-transferase activity) (trans mechanisms)⁵. The observation that distinct PTM patterns cluster at specific chromatin loci led to the elaboration of the hypothesis that different modifications at distinct sites may synergize to generate a molecular code mediating the functional state of the underlying DNA. The "histone code hypothesis" has gained large consensus in the years but its experimental verification has been held back by technical limitations^6,7.

Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to map histone modification patterns and to characterize chromatin-binding proteins⁸. MS detects a modification as a specific Δmass between the experimental and theoretical mass of a peptide. At the level of individual histones, MS provides an unbiased and comprehensive method to map PTMs, allowing the detection of new modifications and revealing interplays among them^9-14.

In recent years, a number of strategies have been developed to dissect the proteomic composition of chromatin, including the characterization of intact mitotic chromosomes¹⁵, the identification of soluble hPTM-binding proteins^16-18 and the isolation and analysis of specific chromatin regions (i.e. telomeres)^19,20. However, the investigation of the locus-specific synergies between histone PTMs, variants, and chromatin-associated proteins is still incomplete. Here, we describe a new approach, named ChroP (Chromatin Proteomics)²¹, that we have developed to efficiently characterize functionally distinct chromatin domains. This approach adapts chromatin immunoprecipitation (ChIP), a well-established protocol used in epigenetic research, for the efficient MS-based proteomic analysis of the enriched sample. We have developed two different protocols, depending on the type of chromatin used as input and the question addressed by MS; in particular: 1) ChIP of unfixed native chromatin digested with MNase is employed to purify mono-nucleosomes and to dissect the co-associating hPTMs (N-ChroP); 2) ChIP of crosslinked chromatin fragmented by sonication is used in combination with a SILAC-based interactomics strategy to characterize all co-enriching chromatin binding proteins (X-ChroP). We illustrate here the combination of N- and X-ChroP for enriching and studying heterochromatin, using H3K9me3 as bait for the immunoprecipitation steps. The use of ChroP can be extended to study either distinct regions on chromatin, or changes in the chromatin composition within the same region during the transition to a different functional state, thus paving the way to various applications in epigenetics.

Protocol

1. Cell Culture Standard medium for native ChIP Grow HeLa cells in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 1% Glutamine, 1% Pen/Strep and 10 mM HEPES pH 7.5. SILAC labelling for crosslinking ChIP Grow HeLa cells in SILAC DMEM medium, depleted of lysine and arginine, supplemented with 10% dialyzed FBS, 1% Glutamine, 1% Pen/Strep, 10 mM HEPES pH 7.5 and either the light L-l…

Representative Results

Chromatin immunoprecipitation is a powerful technique used to profile the localization of a protein or a histone modification along the genome. In a proteomics equivalent, ChIP is followed by MS-based proteomics to identify qualitatively and quantitatively the hPTMs, histone variants and chromatin-binding proteins that are immunoprecipitated together with the modification or protein of interest, used as “bait”. In the N-ChroP approach, outlined in Figure 1A, native ChIP, in which chromatin is…

Discussion

We have recently described ChroP, a quantitative strategy for the large-scale characterization of the protein components of chromatin. ChroP combines two complementary approaches used in the epigenetic field, ChIP and MS, profiting from their strengths and overcoming their respective limitations. ChIP coupled to deep sequencing (ChIP-Seq) allows the genome-wide mapping of histone modifications at the resolution of few nucleosomes³⁵. Although advantageous for their sensitivity, antibody-based assays are limited…

Materials

DMEM	Lonza	BE12-614F
FBS	Invitrogen	10270-106
SILAC DMEM	M-Medical	FA30E15086
Dialyzed FBS	Invitrogen	26400-044
Lysine 0 (12C6 14N2 L-lysine)	Sigma Aldrich	L8662
Arginine 0 (12C6 14N4 L-arginine)	Sigma Aldrich	A6969
Lysine 8 (13C6 15N2 L-lysine)	Sigma Aldrich	68041
Arginine 10 (13C6 15N4 L-arginine)	Sigma Aldrich	608033
Micrococcal Nuclease	Roche	10 107 921 001
Complete, EDTA-free Protease Inhibitor Cocktail Tablets	Roche	04 693 132 001
Spectra/Por 3 dialysis tubing, 3.5K MWCO, 18mm flat width, 50 foot length	Spectrumlabs	132720
QIAquick PCR purification kit	QIAGEN	28104
Anti-Histone H3 tri-methylated K9-ChIP grade	Abcam	ab8898
Histone H3 peptide tri-methyl K9	Abcam	ab1773
Dynabeads Protein G	Invitrogen	100.04D
NuPAGE Novex 4-12%                            Bis-Tris Gel	Invitrogen	NP0335BOX
Colloidal Blue Staining Kit	Invitrogen	LC6025
LDS Sample Buffer	Invitrogen	NP0007
Formaldheyde	Sigma Aldrich	F8775
Aceti anhydride-d6	Sigma Aldrich	175641-1G
Formic Acid	Sigma Aldrich	94318-50ML-F
Iodoacetamide ≥99% (HPLC), crystalline	Sigma Aldrich	I6125
DL-Dithiothreitol	Sigma Aldrich	43815
Sequencing Grade Modified Trypsin, Frozen 100ug (5 × 20μg)	Promega	V5113
Nanospray OD 360μm x ID 75μm, tips ID 8μm uncoated Pk 5	Microcolumn Srl	FS360-75-8-N-5-C15
ReproSil-Pur 120 C18-AQ, 3 µm   15 % C	Dr. Maisch GmbH	r13.aq.
C18 extraction disk, 47 mm	Agilent Technologies	1​2​1​4​5​0​0​4
Carbon extraction disk, 47 mm	Agilent Technologies	12145040
Cation extraction disk	Agilent Technologies	66889-U