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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.


Related in: MedlinePlus

ChIP-MS based methods to identify chromatin associated protein complexes. Cells are crosslinked by formaldehyde and chromatin is prepared by sonication or nuclease digest. Following the appropriate enrichment step, proteins are quantitatively analysed. (A) ChroP (44,45), ChIP-MS (51,52): Chromatin complexes marked with histone PTMs are enriched with PTM-specific antibodies to identify proteins associated with specific chromatin regions (B) mChIP (46,47), ChIP-MS (42,43), RIME (48–50), BioTAP-XL (53,55): Transcription factors or other chromatin associated proteins are targeted via protein tags or antibodies directed towards the endogenous proteins to identify their chromatin-bound protein complexes. Note that in case of mChIP, chromatin is not crosslinked with formaldehyde, hence the method represents a native ChIP-MS protocol. (C) ChIRP-MS (60): lncRNAs associated with chromatin complexes are enriched using an array of biotinylated antisense oligonucleotides spanning the entire sequence of the lncRNA (D) Locus-specific ChIP-MS: an individual genomic locus is enriched using a complementary DNA probe [PICh (79,80)], an engineered artificial binding site together with a respective binding protein - e.g. LexA [ChAP-MS (81), iCHIP (82)] or TetR [TChP (83)] -, or proteins that directly recognize the specific target sequence - e.g. TAL [TAL-ChAP-MS (85), enChIP (86)] or Cas9-sgRNA [CRISPR-ChAP-MS (90), enCHIP (91)]. The binding protein has one or more tags that can be used for purification. (E) The relative bait abundance and amount of input material required for the different ChIP-MS based methods shown in (A-D).
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ddw208-F2: ChIP-MS based methods to identify chromatin associated protein complexes. Cells are crosslinked by formaldehyde and chromatin is prepared by sonication or nuclease digest. Following the appropriate enrichment step, proteins are quantitatively analysed. (A) ChroP (44,45), ChIP-MS (51,52): Chromatin complexes marked with histone PTMs are enriched with PTM-specific antibodies to identify proteins associated with specific chromatin regions (B) mChIP (46,47), ChIP-MS (42,43), RIME (48–50), BioTAP-XL (53,55): Transcription factors or other chromatin associated proteins are targeted via protein tags or antibodies directed towards the endogenous proteins to identify their chromatin-bound protein complexes. Note that in case of mChIP, chromatin is not crosslinked with formaldehyde, hence the method represents a native ChIP-MS protocol. (C) ChIRP-MS (60): lncRNAs associated with chromatin complexes are enriched using an array of biotinylated antisense oligonucleotides spanning the entire sequence of the lncRNA (D) Locus-specific ChIP-MS: an individual genomic locus is enriched using a complementary DNA probe [PICh (79,80)], an engineered artificial binding site together with a respective binding protein - e.g. LexA [ChAP-MS (81), iCHIP (82)] or TetR [TChP (83)] -, or proteins that directly recognize the specific target sequence - e.g. TAL [TAL-ChAP-MS (85), enChIP (86)] or Cas9-sgRNA [CRISPR-ChAP-MS (90), enCHIP (91)]. The binding protein has one or more tags that can be used for purification. (E) The relative bait abundance and amount of input material required for the different ChIP-MS based methods shown in (A-D).

Mentions: Binding of proteins to chromatin in vivo is influenced by many different factors, such as the combinatorics of different modifications on histones and DNA, the presence of DNA-binding proteins other than histone core particles and the three dimensional structure of chromatin (39). In the classical chromatin-immunoprecipitation (ChIP) method chromatin is crosslinked by formaldehyde and sheared by nuclease treatment or sonication to a length of one to three nucleosomes, followed by antibody based affinity purification and analysis of the bound DNA by PCR or next generation sequencing (ChIP-seq) (40,41). Technological improvements in MS have recently allowed the application of these ChIP protocols to elucidate chromatin associated protein complexes, a technology termed ChIP-MS (42,43), ChroP (44,45), mChIP (46,47), or RIME (48–50) (Figure 2A). Two recent papers enriched nucleosomes containing particular histone modifications to study protein interaction networks of genomic regions marked with specific histone PTMs for promoter, enhancer and heterochromatin regions in mouse ESCs (51,52). Interestingly, the same method can also identify co-occurrence of histone marks, by analysing the histone PTM levels in the precipitated material (44). The application of ChIP-MS to histone variants elucidated nucleosomal ratios and histone PTMs of mononucleosomes containing non-canonical histone variants (43).Figure 2.


Proteomics to study DNA-bound and chromatin-associated gene regulatory complexes
ChIP-MS based methods to identify chromatin associated protein complexes. Cells are crosslinked by formaldehyde and chromatin is prepared by sonication or nuclease digest. Following the appropriate enrichment step, proteins are quantitatively analysed. (A) ChroP (44,45), ChIP-MS (51,52): Chromatin complexes marked with histone PTMs are enriched with PTM-specific antibodies to identify proteins associated with specific chromatin regions (B) mChIP (46,47), ChIP-MS (42,43), RIME (48–50), BioTAP-XL (53,55): Transcription factors or other chromatin associated proteins are targeted via protein tags or antibodies directed towards the endogenous proteins to identify their chromatin-bound protein complexes. Note that in case of mChIP, chromatin is not crosslinked with formaldehyde, hence the method represents a native ChIP-MS protocol. (C) ChIRP-MS (60): lncRNAs associated with chromatin complexes are enriched using an array of biotinylated antisense oligonucleotides spanning the entire sequence of the lncRNA (D) Locus-specific ChIP-MS: an individual genomic locus is enriched using a complementary DNA probe [PICh (79,80)], an engineered artificial binding site together with a respective binding protein - e.g. LexA [ChAP-MS (81), iCHIP (82)] or TetR [TChP (83)] -, or proteins that directly recognize the specific target sequence - e.g. TAL [TAL-ChAP-MS (85), enChIP (86)] or Cas9-sgRNA [CRISPR-ChAP-MS (90), enCHIP (91)]. The binding protein has one or more tags that can be used for purification. (E) The relative bait abundance and amount of input material required for the different ChIP-MS based methods shown in (A-D).
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ddw208-F2: ChIP-MS based methods to identify chromatin associated protein complexes. Cells are crosslinked by formaldehyde and chromatin is prepared by sonication or nuclease digest. Following the appropriate enrichment step, proteins are quantitatively analysed. (A) ChroP (44,45), ChIP-MS (51,52): Chromatin complexes marked with histone PTMs are enriched with PTM-specific antibodies to identify proteins associated with specific chromatin regions (B) mChIP (46,47), ChIP-MS (42,43), RIME (48–50), BioTAP-XL (53,55): Transcription factors or other chromatin associated proteins are targeted via protein tags or antibodies directed towards the endogenous proteins to identify their chromatin-bound protein complexes. Note that in case of mChIP, chromatin is not crosslinked with formaldehyde, hence the method represents a native ChIP-MS protocol. (C) ChIRP-MS (60): lncRNAs associated with chromatin complexes are enriched using an array of biotinylated antisense oligonucleotides spanning the entire sequence of the lncRNA (D) Locus-specific ChIP-MS: an individual genomic locus is enriched using a complementary DNA probe [PICh (79,80)], an engineered artificial binding site together with a respective binding protein - e.g. LexA [ChAP-MS (81), iCHIP (82)] or TetR [TChP (83)] -, or proteins that directly recognize the specific target sequence - e.g. TAL [TAL-ChAP-MS (85), enChIP (86)] or Cas9-sgRNA [CRISPR-ChAP-MS (90), enCHIP (91)]. The binding protein has one or more tags that can be used for purification. (E) The relative bait abundance and amount of input material required for the different ChIP-MS based methods shown in (A-D).
Mentions: Binding of proteins to chromatin in vivo is influenced by many different factors, such as the combinatorics of different modifications on histones and DNA, the presence of DNA-binding proteins other than histone core particles and the three dimensional structure of chromatin (39). In the classical chromatin-immunoprecipitation (ChIP) method chromatin is crosslinked by formaldehyde and sheared by nuclease treatment or sonication to a length of one to three nucleosomes, followed by antibody based affinity purification and analysis of the bound DNA by PCR or next generation sequencing (ChIP-seq) (40,41). Technological improvements in MS have recently allowed the application of these ChIP protocols to elucidate chromatin associated protein complexes, a technology termed ChIP-MS (42,43), ChroP (44,45), mChIP (46,47), or RIME (48–50) (Figure 2A). Two recent papers enriched nucleosomes containing particular histone modifications to study protein interaction networks of genomic regions marked with specific histone PTMs for promoter, enhancer and heterochromatin regions in mouse ESCs (51,52). Interestingly, the same method can also identify co-occurrence of histone marks, by analysing the histone PTM levels in the precipitated material (44). The application of ChIP-MS to histone variants elucidated nucleosomal ratios and histone PTMs of mononucleosomes containing non-canonical histone variants (43).Figure 2.

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