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Proteomics to study DNA-bound and chromatin-associated gene regulatory complexes

View Article: PubMed Central - PubMed

ABSTRACT

High-resolution mass spectrometry (MS)-based proteomics is a powerful method for the identification of soluble protein complexes and large-scale affinity purification screens can decode entire protein interaction networks. In contrast, protein complexes residing on chromatin have been much more challenging, because they are difficult to purify and often of very low abundance. However, this is changing due to recent methodological and technological advances in proteomics. Proteins interacting with chromatin marks can directly be identified by pulldowns with synthesized histone tails containing posttranslational modifications (PTMs). Similarly, pulldowns with DNA baits harbouring single nucleotide polymorphisms or DNA modifications reveal the impact of those DNA alterations on the recruitment of transcription factors. Accurate quantitation – either isotope-based or label free – unambiguously pinpoints proteins that are significantly enriched over control pulldowns. In addition, protocols that combine classical chromatin immunoprecipitation (ChIP) methods with mass spectrometry (ChIP-MS) target gene regulatory complexes in their in-vivo context. Similar to classical ChIP, cells are crosslinked with formaldehyde and chromatin sheared by sonication or nuclease digested. ChIP-MS baits can be proteins in tagged or endogenous form, histone PTMs, or lncRNAs. Locus-specific ChIP-MS methods would allow direct purification of a single genomic locus and the proteins associated with it. There, loci can be targeted either by artificial DNA-binding sites and corresponding binding proteins or via proteins with sequence specificity such as TAL or nuclease deficient Cas9 in combination with a specific guide RNA. We predict that advances in MS technology will soon make such approaches generally applicable tools in epigenetics.

No MeSH data available.


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In vitro pulldown assays to study protein interactions with modified histone tails, DNA and assembled nucleosomes. Nuclear cell extracts are incubated with (A) peptides resembling a partial histone tail, which can carry posttranslational modifications, (B) in vitro assembled nucleosomes, harboring a DNA modification and/or modified histone tail integrated by chemical linkage, or (C) DNA baits, which harbor a specific DNA sequence, such as a disease-linked SNP or a DNA modification. Following enrichment, proteins are enzymatically digested and analysed by high resolution LC-MS/MS. Specific interactors are defined by quantitative comparison with a control by means of isotope or label-free quantitation-based methods.
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ddw208-F1: In vitro pulldown assays to study protein interactions with modified histone tails, DNA and assembled nucleosomes. Nuclear cell extracts are incubated with (A) peptides resembling a partial histone tail, which can carry posttranslational modifications, (B) in vitro assembled nucleosomes, harboring a DNA modification and/or modified histone tail integrated by chemical linkage, or (C) DNA baits, which harbor a specific DNA sequence, such as a disease-linked SNP or a DNA modification. Following enrichment, proteins are enzymatically digested and analysed by high resolution LC-MS/MS. Specific interactors are defined by quantitative comparison with a control by means of isotope or label-free quantitation-based methods.

Mentions: Typically, peptides representing parts of the histone tail sequence and containing one or more PTMs are linked with beads and incubated with cell extracts. Proteins specifically recognizing the PTM are enriched and then analysed by MS (Figure 1A) . Such assays identified key events such as the binding of Wdr5 and NURF to trimethylated lysine 4 on histone H3 (29,30). The introduction of quantitative proteomics in the form of SILAC labeling (4) dramatically increased sensitivity and specificity, revealing that the TFIID complex directly interacts with H3K4me3 (31) as well as interactions of other factors with trimethylated lysines (32). The same approach elegantly unraveled differential binding of different PRMT isoforms to symmetric and asymmetric dimethylation of H3R2 (33). Improvements in MS data quality and algorithms have led to a switch to label-free quantitation and this has successfully been applied to mouse tissues (34) and even birds (35). To more closely resemble binding in the context of chromatin, mono- or oligo-nucleosomes with modified histone tails have been generated by their chemical linkage to the globular domains (36–38) (Figure 1B). Although the differences in reader proteins in peptide and nucleosome pulldowns for a given histone PTM appear to be minor, an advantage of nucleosome pulldowns is the combined assessment of histone and DNA modifications. For instance, while PRC2 complex members effectively bound H3K27me3 modified nucleosomes, this interaction was lost upon introduction of DNA methylation, while the association with the ORC complex was increased (38).Figure 1.


Proteomics to study DNA-bound and chromatin-associated gene regulatory complexes
In vitro pulldown assays to study protein interactions with modified histone tails, DNA and assembled nucleosomes. Nuclear cell extracts are incubated with (A) peptides resembling a partial histone tail, which can carry posttranslational modifications, (B) in vitro assembled nucleosomes, harboring a DNA modification and/or modified histone tail integrated by chemical linkage, or (C) DNA baits, which harbor a specific DNA sequence, such as a disease-linked SNP or a DNA modification. Following enrichment, proteins are enzymatically digested and analysed by high resolution LC-MS/MS. Specific interactors are defined by quantitative comparison with a control by means of isotope or label-free quantitation-based methods.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036873&req=5

ddw208-F1: In vitro pulldown assays to study protein interactions with modified histone tails, DNA and assembled nucleosomes. Nuclear cell extracts are incubated with (A) peptides resembling a partial histone tail, which can carry posttranslational modifications, (B) in vitro assembled nucleosomes, harboring a DNA modification and/or modified histone tail integrated by chemical linkage, or (C) DNA baits, which harbor a specific DNA sequence, such as a disease-linked SNP or a DNA modification. Following enrichment, proteins are enzymatically digested and analysed by high resolution LC-MS/MS. Specific interactors are defined by quantitative comparison with a control by means of isotope or label-free quantitation-based methods.
Mentions: Typically, peptides representing parts of the histone tail sequence and containing one or more PTMs are linked with beads and incubated with cell extracts. Proteins specifically recognizing the PTM are enriched and then analysed by MS (Figure 1A) . Such assays identified key events such as the binding of Wdr5 and NURF to trimethylated lysine 4 on histone H3 (29,30). The introduction of quantitative proteomics in the form of SILAC labeling (4) dramatically increased sensitivity and specificity, revealing that the TFIID complex directly interacts with H3K4me3 (31) as well as interactions of other factors with trimethylated lysines (32). The same approach elegantly unraveled differential binding of different PRMT isoforms to symmetric and asymmetric dimethylation of H3R2 (33). Improvements in MS data quality and algorithms have led to a switch to label-free quantitation and this has successfully been applied to mouse tissues (34) and even birds (35). To more closely resemble binding in the context of chromatin, mono- or oligo-nucleosomes with modified histone tails have been generated by their chemical linkage to the globular domains (36–38) (Figure 1B). Although the differences in reader proteins in peptide and nucleosome pulldowns for a given histone PTM appear to be minor, an advantage of nucleosome pulldowns is the combined assessment of histone and DNA modifications. For instance, while PRC2 complex members effectively bound H3K27me3 modified nucleosomes, this interaction was lost upon introduction of DNA methylation, while the association with the ORC complex was increased (38).Figure 1.

View Article: PubMed Central - PubMed

ABSTRACT

High-resolution mass spectrometry (MS)-based proteomics is a powerful method for the identification of soluble protein complexes and large-scale affinity purification screens can decode entire protein interaction networks. In contrast, protein complexes residing on chromatin have been much more challenging, because they are difficult to purify and often of very low abundance. However, this is changing due to recent methodological and technological advances in proteomics. Proteins interacting with chromatin marks can directly be identified by pulldowns with synthesized histone tails containing posttranslational modifications (PTMs). Similarly, pulldowns with DNA baits harbouring single nucleotide polymorphisms or DNA modifications reveal the impact of those DNA alterations on the recruitment of transcription factors. Accurate quantitation – either isotope-based or label free – unambiguously pinpoints proteins that are significantly enriched over control pulldowns. In addition, protocols that combine classical chromatin immunoprecipitation (ChIP) methods with mass spectrometry (ChIP-MS) target gene regulatory complexes in their in-vivo context. Similar to classical ChIP, cells are crosslinked with formaldehyde and chromatin sheared by sonication or nuclease digested. ChIP-MS baits can be proteins in tagged or endogenous form, histone PTMs, or lncRNAs. Locus-specific ChIP-MS methods would allow direct purification of a single genomic locus and the proteins associated with it. There, loci can be targeted either by artificial DNA-binding sites and corresponding binding proteins or via proteins with sequence specificity such as TAL or nuclease deficient Cas9 in combination with a specific guide RNA. We predict that advances in MS technology will soon make such approaches generally applicable tools in epigenetics.

No MeSH data available.


Related in: MedlinePlus