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.
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.
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 wrapped1,2. 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 histones3,4. 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 limitations6,7.
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 them9-14.
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 proteins16-18 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.
1. Cell Culture
2. Native Chromatin Immunoprecipitation (N-ChIP)
3. Crosslinking Chromatin Immunoprecipitation (X-ChIP)
4. Sample Preparation Prior to MS
5. LC-MS Analysis
6. Data Analysis
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 digested with MNase (Figure 1B), is used as input to purify from bulk chromatin a distinct functional domain. Digested chromatin enriched in mono-nucleosomes is incubated with a specific antibody and the immunopurified proteins are separated by SDS-PAGE. In the example illustrated, H3K9me3, marker of silent chromatin32,33, is used to set up the approach. The choice is based on the fact that both its functional role as well as some of its protein interactors are well described. Moreover, a highly specific and efficient antibody optimized for ChIP is available34, as also confirmed by visual inspection of the Coomassie gel, where the intact nucleosome, with the core histones at the correct stoichiometry, is enriched in appropriate amounts for MS (Figure 1D).
The comparison between the amount of H3K9me3 present in the flow-through (FT) and input (IN) indicates that about 50% of the region of interest is immunopurified, excluding the risk of a bias due the enrichment of a minor sub-population of chromatin (Figure 1C). MS is employed to characterize the PTMs co-associated within the enriched nucleosomes: each core histone is digested using a protocol designed ad hoc in order to achieve an “Arg-C like” digestion, in polyacrylamide gel. In fact, on the one hand Arg-C is the best protease for MS analysis of hPTMs because it produces peptides of optimal length; on the other hand, it does not cleave efficiently in gel. To overcome this limitation, our tailored protocol exploits the chemical alkylation of lysine achieved by incubating histone gel bands with deuterated (D6)-acetic anhydride, followed by trypsin digestion. Since trypsin does not cleave D3-acetylated lysines, the resulting peptides mixture mimics an “Arg-C like” pattern (Figure 1E).
The addition of a D3-acetyl moiety to a lysine produces an unambiguous delta mass of 45.0294 Daltons, discriminating between native and chemically added acetylations in MS. Moreover, D3-acetylation offers two additional advantages that facilitate the discernment of isobaric modified peptides: first, the alkylation occurs only on unmodified and mono-methylated lysines, but not on di- and tri-methylated residues; as such modified peptides bearing the same total number of modifications but in different arrangements are differentially decorated by distinct set of D3-acetyl groups that produce unambiguous m/z shifts. Second, distinct patterns of D3-alkylation cause slightly different retention times in liquid chromatography on reverse phase column, which generate an additional level of separation for isobaric modified peptides21.
After validation of all hPTMs by manual inspection of the corresponding MS/MS spectra (Figure 2B), the label free quantification is achieved in two steps: first, we calculate the relative abundance of each modification using the signal intensity of the unmodified and modified species for the corresponding peptide, measured through the calculation of the extracted ion chromatograms (XIC) (Figure 2A and 2C, upper panel); second, the relative enrichment is estimated as a ratio between the relative abundance of each modification in the ChIP-ed octamer and the corresponding relative abundance from input (Figure 2C, lower panel). The analysis of the H3(9-17) peptide shows the enrichment of di- and tri-methylated K9, with the corresponding depletion of the unmodified and mono-methylated forms (Figure 2C). With this results as positive control for antibody specificity, the co-association or depletion of all other modifications can be assessed, both at the intra-molecular level on the same H3 and at the inter-molecular level, on other co-enriched histones within the same nucleosome. This allows the screening of hPTMs cross-talks within the H3K9me3 domains, the so-called “heterochromatin modificome” (Figure 2D): the significant enrichment of known markers associated with gene silencing, with the corresponding depletion of markers linked with gene activation is observed. In addition, novel associations are detected such as the enrichment of H3K18me1.
To screen for all proteins co-associating within heterochromatin, the classical crosslinking ChIP is combined with SILAC (Stable Isotope Labelling by Amino acids in Cell culture) (Figure 3A). In the SILAC experiment, the lysine and arginine (light form) are replaced with their isotopically labeled analogs (heavy form) in the culturing medium. Upon cells grown and replication in both light and heavy media, the two differentially isotope-encoded amino acids are metabolically incorporated into proteins, generating light and heavy forms of proteins, respectively, that are distinguishable by MS, due to a specific Δmass. Before starting a large-scale SILAC experiment, the labeling efficiency is evaluated, calculating the incorporation level, measured as the percentage fraction of heavy peptides versus the sum of heavy and light ones, found in the only heavy labeled sample. Incorporation superior to 95% for both heavy arginine and heavy lysine is required for accurate protein quantification (Figure 3B). After crosslinking of heavy and light cells, chromatin is fragmented by sonication. SILAC is used in conjunction with a competition assay using an excess fold of soluble H3 peptide (QTARKSTGG) that bears tri-methylation on K9 in order to discriminate specific H3K9me3 interactors from background. The soluble peptide is added in excess to one of the two SILAC ChIP experiments, where it saturates the binding capacity of the antibody, thus “competing out” the majority of the H3K9me3- nucleosomes and, accordingly, all specific interactors. In the other SILAC channel, the competing peptide is not added to the ChIP and the immunoprecipitation takes place normally. SILAC-competition experiments are typically performed in duplicate in so-called “Forward” and “Reverse” formats, where the competition with the excess soluble peptide is switched from the heavy (H) to the light (L) chromatin samples. This results in inverted complementary SILAC ratio readouts, used for discerning genuine from unspecific binders: proteins specifically enriched are present with a higher intensity in the heavy form in comparison with the light form (protein ratio H/L>1) in the experiment where excess peptide is added to the light channel (Forward) (Figure 4A, upper panel); an opposite trend (protein ratio H/L<1) is observed in the Reverse replica (Figure 4A, lower panel). Proteins whose intensity in the heavy and light form are similar in both Forward and Reverse experiments produce a constant ratio close to 1 and are classified as background (Figure 4B).
The choice of the optimal excess fold for the soluble peptide is important and must be tuned accurately. Typically, we set the correct antibody-to-peptide proportion performing a competition assay using serial dilutions of excess peptide and comparing the level of the “bait” (hPTM/protein) between the positive control ChIP, where the peptide is not added, and the different serial competition ChIPs, by Western Blot or MS. Generally, the optimal excess fold peptide reduces of about 90% the amount of hPTM/protein in the immunoprecipitated material. In fact, on the one hand, the larger the protein ratio obtained, the higher the discriminating power of SILAC; on the other hand, an excessive saturation by the peptide may evict completely the specific binders from the antibody, thus leading to missing H/L ratios for quantification and statistical analysis. For H3K9me3 antibody we defined the correct proportion by measuring the H/L ratio for the QTARK(me3)STGG peptide, in a competition test carried out in a Forward set up and we took this value as a measure of the competition efficiency (Figure 3C).
The intersection of the Forward and Reverse X-ChIP experiments leads to the identification of 635 proteins, present in both experiments and quantified with at least 2 ratio counts. The log2 plot of their H/L ratios represent the so-called “heterochromatome” (Figure 4C), where the genuine H3K9me3 interactors are unambiguously identified as the proteins present within the top 40% of the proteins ratio distributions (upper right quadrant of the scatter plot and Figure 4D).
Figure 1: Schematic view of the N-ChroP workflow. A) Scheme of native ChIP combined with MS analysis. Chromatin from cells is digested with MNase and the fraction enriched in mono-nucleosomes (S1) is immunoprecipitated using an anti-H3K9me3 antibody. Immunopurified proteins are separated by SDS-PAGE and core histones are in gel-digested with an ad hoc protocol to mimic an Arg-C digestion. Peptides are analyzed by nano-LC-MS/MS. Histone PTMs are identified, validated by manual inspection of MS/MS spectra and quantified. B) Small scale MNase test: DNA resolved on a 1% agarose gel after chromatin digestion with MNase at different time lapse (left panel); large scale MNase digestion: DNA resolved on a 1% agarose gel after 60 minutes of MNase chromatin digestion and after separation of S1 fraction, containing mono-nucleosomes from S2 faction, containing poly-nucleosomes (right panel). C) Estimation of enrichment/depletion of unmodified, mono-, di-, and tri-methylated K9 in flow-through (FT) compared to input (IN). Histogram represents the average ±SEM from three independent experiments for each modification. D) SDS-PAGE of chromatin input and co-immunopurified proteins: core histones H3, H4, H2A and H2B are visible around and below the 17kDa band, with H3 and H2B co-migrating (black squares). E) Each core histone from both immunoprecipitated nucleosomes and input are chemically alkylated using deuterated (D6)-acetic-anhydride before trypsin treatment, in order to obtain an “Arg-C like” in gel digestion. The D6-acetic anhydride reacts with the Epsilon amino group of unmodified and mono-methylated lysines but not with di-methylated, tri-methylated and acetylated lysines. As result, the enzymatic activity of trypsin is blocked on all native and chemical acetylated lysine, thus producing a “Arg-C like” digestion pattern. This figure has been modified from 21, using Figure 1, Figure S1 and Figure S5 as reference. Click here to view larger image.
Figure 2: Mass Spectrometry analysis of the H3K9me3 “modificome”. A) Zoomed mass spectra and extracted ion chromatograms (XIC) constructed at the corresponding m/z value of the 2+ charge unmodified, mono-, di-, and tri-methylated K9 in the H3(9-17) peptide, both for input and ChIP samples. B) Representative MS/MS spectra using CID fragmentation. The b-ion and y-ion series allow to define the sequence of H3(9-17) peptide and to localize specifically the tri-methylation on K9 residue. C) Relative abundance of the different degrees of methylation on K9 in the H3(9-17) peptide, estimated by dividing the area under the curve (AUC) of each modified peptide by the sum of the areas corresponding to all observed unmodified and modified forms of that peptide, in the input and ChIP-ed octamer. Histogram represents the average ±SEM from three independent experiments for each modification (upper panel). Relative enrichment of K9 methylations in H3(9-17) peptide. The enrichment is expressed as a log2 ratio between the relative abundance of each methylation in the ChIP-ed octamer as compare to input. Histogram represents the averages ±SEM from three independent experiments (lower panel). D) Heatmap summarizing the enrichment of all co-associating hPTMs identified on histone H3, H4 and H2A. Each row corresponds to a different modification (n.d. are not detected modifications). This figure has been modified from 21, using Figure 1, Figure 2, Figure 3 and Figure S10 as reference. Click here to view larger image.
Figure 3: Schematic view of X-ChroP workflow. A) Scheme of crosslinking ChIP combined with MS analysis. Cells grown in light and heavy media are fixed with formaldehyde and chromatin inputs are fragmented by sonication to generate DNA fragments. In a Forward set up, the sonicated heavy-labeled chromatin is immunoprecipitated using anti-H3K9me3 antibody while the light-labeled chromatin is incubated with the same antibody saturated with an excess fold of soluble H3 peptide bearing K9me3. Immunoprecipitated proteins from heavy and light chromatins are pooled, extracted and separated by SDS-PAGE. Proteins are digested with trypsin and peptides are analyzed by nano-LC-MS/MS. B) Efficiency of SILAC labeling is monitored calculating the incorporation of heavy lysine (Lys8) and arginine (Arg10) into proteins. In the plot, the median of lysine and arginine peptides density distribution is equal to 0.974 (green line) and 0.964 (red line), respectively. C) Zoomed mass spectra at the corresponding m/z value of the 2+ charge tri-methylated K9 in the H3(9-17) peptide, both for light and heavy forms, in input and ChIP-ed octamer. While in input the intensities of heavy and light peptide are close to 1, in the Forward SILAC ChIP, the intensity of light peptide is much lower than the one of the heavy counterpart, thus indicating an efficient competition. D) SDS-PAGE of light (L) and heavy (H) labeled chromatin input and of co-immunoprecipitated material: the lane corresponding to the ChIP-ed material is cut in ten slices (black line), while only two slices of input are analyzed for incorporation test analysis (blue line). This figure has been modified from 21, using Figure 4 and Figure S6 as reference. Click here to view larger image.
Figure 4: Mass Spectrometry analysis of the H3K9me3 “interactome”. A) Representative full spectra showing the SILAC-pair corresponding to peptides from HP1 and macro-2A proteins: the ratios H/L>1 in the Forward experiment (upper panels), mirrored by a ratios H/L<1 in the Reverse replica (lower panels), demonstrate the specific enrichment of these proteins in heterochromatin. B) Representative full spectra with SILAC-pair corresponding to peptide from one background protein: H/L ratios in Forward and Reverse replicates are equal to 1. C) Quantified proteins are distributed in a scatter plot based on their SILAC-ratios in Forward and Reverse experiments (x and y axes, respectively); red dotted lines represent the top 40% and 30% of protein ratios, as indicated. D) Protein ratios distributions from input (H/L mixed 1:1) (black), Forward (blue) and Reverse (orange) X-ChroP experiments; red dotted lines represent the top 40% of protein ratios, set as cutoffs to select the genuine interactors. This figure has been modified from21, using Figure 5 as reference. Click here to view larger image.
Buffer for section 2 | Composition |
Lysis Buffer | 10% sucrose, 0.5 mM EGTA pH 8.0, 15 mM NaCl, 60 mM KCl, 15 mM HEPES, 0.5% Triton, 0.5 mM PMSF, 1mM DTT, 5 mM NAF, 5 mM Na3VO4, 5mM NaButyrate, 5 mg/ml Aprotinin, 5 mg/ml Pepstatin A, 5 mg/ml Leupeptin |
Sucrose cushion | 2 g sucrose in 20 ml of Lysis Buffer |
Digestion Buffer | 0.32 M sucrose, 50 mM Tris-HCl pH 7.6, 4 mM MgCl2, 1 mM CaCl2, 0.1 mM PMSF |
TE Buffer | 10 mM Tris-HCl pH 7.5, 1 mM EDTA |
Dialysis Buffer | 10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.5 mM PMSF, 5 mM NAF, 5 mM Na3VO4, 5mM NaButyrate, protease inhibitors cocktail |
ChIP Dilution Buffer | 100 mM Tris HCl pH 7.6, 100 mM NaCl and 10 mM EDTA |
Blocking Solution | BSA 0.5% in PBS |
Washing Buffer | 50 mM Tris-HCl pH 7.6,10 mM EDTA |
Protease Inhibitors | EDTA-free protease inhibitors cocktail, 0.5 mM PMSF, 5 mM NAF, 5 mM Na3VO4, 5mM NaButyrate |
Loading Buffer | orange loading dye, 50% (v/v) glycerol in H20 |
Buffer for section 3 | Composition |
Lysis Buffer | 50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton-100, 0.5 mM PMSF, 5 mM NAF, 5 mM Na3VO4, 5mM NaButyrate, 5 mg/ml Aprotinin, 5 mg/ml Pepstatin A, 5 mg/ml Leupeptin |
Washing Buffer | 10 mM Tris-HCl pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.5 mM PMSF, 5 mM NAF, 5 mM Na3VO4, 5mM NaButyrate, 5 mg/ml Aprotinin, 5 mg/ml Pepstatin A, 5 mg/ml Leupeptin |
ChIP Incubation Buffer | 10 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% sodium lauroylsarcoside, 0.5 mM PMSF, 5 mM NAF, 5 mM Na3VO4, 5 mM NaButyrate, 5 mg/ml Aprotinin, 5 mg/ml Pepstatin A, 5 mg/ml Leupeptin |
Washing Buffer 2 | 20 mM Tris-HCl pH 7.6, 2 mM EDTA, 0.1 % SDS,1 % Triton-100 |
Loading Sample Buffer | 250 mM Tri-HCl pH 8.8, 0.5 M b-mercaptoethanol, 2% SDS |
Buffer for section 4 | Composition |
Alkylation Buffer | 55 mM iodoacetamide in 50 mM NH4HCO3 |
Reduction Buffer | 10 mM dithiothreitol in 50 mM NH4HCO3 |
Extraction Buffer | 30% ACN and 3% TFA in ddH20 |
Table 1. Buffers Composition.
Tuning acquisition file | Parameters |
FT full scan | accumulation target value 1 x 106; maximum filling time 1,000 msec |
IT MSn | accumulation target value 10 x 104; maximum filling time 150 msec |
Acquisition setting | Parameters |
Electrospray voltage | 2.4 kV |
Sheath and auxiliary gas flow | No |
Ion transfer capillary temperature | 200 °C |
Dynamic exclusion | up to 500 precursor ions for 60 sec upon MSMS |
Exclusion mass width | 10 ppm |
Normalized collision energy using wide-band activation mode | 35% |
Ion selection thresholds | 100 counts |
Activation q | 0.25 |
Activation time | 30 msec |
Table 2. MS settings.
Mascor Deamon search | Parameters | Notes |
Database | depends on the organism (i.e. for HeLa cells: human database, version 3.68; 87,061 entries) | |
Enzyme | Arg-C | Arg-C cleave at the C-terminal of all arginine residues |
Variable modifications | acetyl (K), oxidation (M), D3-acetylation (K), methyl-D3-acetyl (K), dimethyl (k), trimethyl (K) | acetyl [42.010 Da], oxidation [15.995 Da], D3-acetylation [+45.0294 Da], methyl-D3-acetyl [sum of +14.016 Da and +45.0294 Da], dimethyl [28.031], trimethyl [42.046 Da] |
Missed cleavages | up to 2 | |
Mass accuracy of the parent ions in the search | 10 ppm | |
Mass-accuracy for CID MSMS | 0.5 Da | |
MaxQuant search | Parameters | Notes |
Database | depends on the organism | |
Enzyme | trypsin/P | Trypsin cleaves at the C-terminal of all lysine and arginine residues. The search is performed taking in account the fact that the efficiency of trypsin to cleave lysine and arginine is reduced when the next amino acid is the proline (/P). |
Fixed modification | carbamidomethylation | |
Variable modifications | N-acetyl (Protein), oxidation (M) | |
Missed cleavages | up to 3 | |
Label parameters | lys8 and arg10 | |
Maximum label amminoacid | 3 for Trypsin | |
Mass-accuracy of the parent ions in the initial “Andromeda” search | 20 ppm | |
Mass accuracy of the parent ions in the main “Andromeda” search | 6 ppm | |
Mass-accuracy for CID MSMS | 0.5 Da (six top peaks per100 Da) | |
Peptide false discovery rates (FDR) | 0.01 | |
Protein false discovery rates (FDR) | 0.01 | Setting the FDR for peptide and protein to 0.01 means that both peptides and proteins identified are expected to contain 1% of false positives. This value is estimated using a target-decoy database |
Maximum posterior error probability (PEP) | 1 | PEP is the probability that an individual peptide is a false positive match. PEP equal to 1 in your setting means that all peptides will be taken irrespective of the PEP thus filtering is based exclusively on FDR. |
Minimum peptide length | 6 | |
Minimum number of peptides | 2 | |
Minimum number of unique peptides | 1 | |
Using only unmodified peptides and oxidation (M)/acetyl (Protein N-Term) | activate the option | Peptides with modifications should generally not be counted for protein quantification since their abundance may not reflect the ratio of the corresponding protein. |
Minimum ratio count | 1 | |
“Match between runs” | activate the option | This option matches precursor masses in a 2-min retention time window (after realignment of the runs) based on the accurate mass measurement and allows transferring the MS/MS identification between the different LC MS/MS runs. |
Table 3. Data Analysis.
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 in their capability to distinguish similar modifications and in dissecting the combinatorial aspect of the histone code36. On the other hand, while MS provides a comprehensive and unbiased analysis of hPTMs, singly and in combinations9, it has so far been applied to the analysis of bulk chromatin, with consequent lack of information on locus-specific PTMs patters. We employ a modified version of ChIP to isolate functionally distinct chromatin domains and mass spectrometry to characterize the histone PTM patterns and the non-histonic proteins specifically co-enriched.
By using N-ChroP, the analysis of hPTMs co-associated with the H3K9me3 revealed a significant enrichment of markers associated with silent chromatin, and a depletion of modifications associated with active chromatin. The agreement of these results with previous studies37 proved the robustness of the strategy. In X-ChroP of H3K9me3 domains, the SILAC-based investigation of the chromatin-interacting proteins confirmed some previously described interactors, thereby validating the method.
ChroP presents two main original aspects in respect to the strategies already available to investigate the proteomic component of chromatin: for instance, the possibility to reveal inter-molecular synergisms between modifications decorating distinct core histones within the same intact mono-nucleosome purified by N-ChIP; second the chance to assess the specific compartmentalization of histone variants and linker histone subtypes. Since the investigation of histone variants is held back by the lack of good quality reagents (i.e. antibodies), ChroP emerges as the unique tool available to assess their location and functional role.
One limitation of ChroP in its current set up lies in peptide-centric (“Bottom-up”) approach used in MS, with histones digested in short peptides and the consequent impairment in detecting the long-distance connectivity between modifications. As such, the conjugation of ChroP with “Bottom-up” MS analysis permits a partial assessment of the combinatorial aspect of the histone code. We envisage that the implementation of alternative MS strategies, such as “Middle- and Top-Down” for hPTMs mapping on longer peptides (>20 aa) up to on intact proteins38-40, will overcome this restraint.
Overall, N- and X-ChroP are highly complementary, with the possibility of dissecting the complexity of chromatin structure at functionally distinct domains, with a resolution of mono- to oligo-nucleosomes. We predict that ChroP will be useful also to characterize the composition of chromatin regions marked by the presence of specific non-histonic nuclear proteins, for example transcription factors (TFs). In addition, ChroP can be employed in functional studies to map the dynamic composition of chromatin at specific loci upon various perturbations, for instance during global transcriptional activation. For these reasons, ChroP emerges as an additional useful tool in the arsenal of analytical strategies available to dissect the proteomic landscape of chromatin.
The authors have nothing to disclose.
This research was originally published in Mol Cell Proteomics. Soldi M. and Bonaldi T. The Proteomic Investigation of Chromatin Functional Domains Reveals Novel Synergisms among Distinct Heterochromatin Components MCP. 2013; 12: 64-80. © the American Society for Biochemistry and Molecular Biology. We thank Roberta Noberini (Italian Institute of Technology and IEO, Italy) for critical reading of the manuscript. TB work is supported by grants from the Giovanni Armenise-Harvard Foundation Career Development Program, the Italian Association for Cancer Research and the Italian Ministry of Health. MS work was supported by a FIRC fellowship.
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 | 12145004 | |
Carbon extraction disk, 47 mm | Agilent Technologies | 12145040 | |
Cation extraction disk | Agilent Technologies | 66889-U |