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Purification of a specific native genomic locus for proteomic analysis.

Byrum SD, Taverna SD, Tackett AJ - Nucleic Acids Res. (2013)

Bottom Line: Here, we describe an approach to isolate native chromatin sections without genomic engineering for label-free proteomic identification of associated proteins and histone post-translational modifications.The TAL-PrA fusion enabled chromatin affinity purification (ChAP) of a small section of native chromatin upstream from the GAL1 locus, permitting mass spectrometric (MS) identification of proteins and histone post-translational modifications regulating galactose-induced transcription.This TAL-ChAP-MS approach allows the biochemical isolation of a specific native genomic locus for proteomic studies and will provide for unprecedented objective insight into protein and epigenetic mechanisms regulating site-specific chromosome metabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA and Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.

ABSTRACT
Here, we describe an approach to isolate native chromatin sections without genomic engineering for label-free proteomic identification of associated proteins and histone post-translational modifications. A transcription activator-like (TAL) protein A fusion protein was designed to recognize a unique site in the yeast GAL1 promoter. The TAL-PrA fusion enabled chromatin affinity purification (ChAP) of a small section of native chromatin upstream from the GAL1 locus, permitting mass spectrometric (MS) identification of proteins and histone post-translational modifications regulating galactose-induced transcription. This TAL-ChAP-MS approach allows the biochemical isolation of a specific native genomic locus for proteomic studies and will provide for unprecedented objective insight into protein and epigenetic mechanisms regulating site-specific chromosome metabolism.

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TAL proteins can specifically enrich native chromatin sections. (A) Schematic overview of TAL-ChAP-MS technology. (B) A unique DNA sequence in the promoter region of GAL1 was used to design a specific binding TAL protein that contained a PrA affinity tag. (C) A pTAL-PrA plasmid was introduced into S. cerevisiae cells, and the constitutive expression of the TAL-PrA fusion protein was confirmed by western blotting for PrA. (D) Expression of TAL-PrA does not impede galactose-induced GAL1 transcription. cDNA from wild-type yeast and wild-type with a plasmid expressing PrA-tagged TAL (+pTAL-PrA) grown in glucose (Glu) or galactose (Gal) was used as a template for real time PCR analysis of GAL1 versus ACT1 gene transcription. Error bars are the standard deviation. (E) TAL-PrA specifically binds and enriches chromatin at the promoter of transcriptionally active GAL1. ChIP was performed to the PrA-tag in wild-types cells containing the TAL-PrA (+pTAL-PrA, light gray bars) and in wild-type control (dark gray bars). The efficiency of GAL1 enrichment relative to ACT1 was monitored by real-time PCR with primers targeted to the TAL binding site (‘0’) and to DNA sequences 2000 bp up- and downstream. The standard deviation is indicated.
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gkt822-F1: TAL proteins can specifically enrich native chromatin sections. (A) Schematic overview of TAL-ChAP-MS technology. (B) A unique DNA sequence in the promoter region of GAL1 was used to design a specific binding TAL protein that contained a PrA affinity tag. (C) A pTAL-PrA plasmid was introduced into S. cerevisiae cells, and the constitutive expression of the TAL-PrA fusion protein was confirmed by western blotting for PrA. (D) Expression of TAL-PrA does not impede galactose-induced GAL1 transcription. cDNA from wild-type yeast and wild-type with a plasmid expressing PrA-tagged TAL (+pTAL-PrA) grown in glucose (Glu) or galactose (Gal) was used as a template for real time PCR analysis of GAL1 versus ACT1 gene transcription. Error bars are the standard deviation. (E) TAL-PrA specifically binds and enriches chromatin at the promoter of transcriptionally active GAL1. ChIP was performed to the PrA-tag in wild-types cells containing the TAL-PrA (+pTAL-PrA, light gray bars) and in wild-type control (dark gray bars). The efficiency of GAL1 enrichment relative to ACT1 was monitored by real-time PCR with primers targeted to the TAL binding site (‘0’) and to DNA sequences 2000 bp up- and downstream. The standard deviation is indicated.

Mentions: To alleviate genomic engineering for affinity enrichment of chromatin sections, we report the use of modified transcription activator-like (TAL) effector proteins to site-specifically target a native section of a chromosome for purification and proteomic analysis. We term this approach TAL-ChAP-MS (Figure 1A). TAL effector proteins are from Xanthomonas, which infects plants and translocates TAL effectors into cells where they serve as transcription activators (14–16). TALs contain a central domain of 18 tandem repeats of 34 amino acids each, which direct sequence-specific DNA binding (16,17). Binding to a given nucleobase in DNA is determined by two adjacent amino acids (12 and 13) within each of the 18 repeats (14). Thus, by mutating these amino acids in each of the 18 tandem repeats, one can ‘program’ binding to a given 18-nt region of DNA in vivo. TAL proteins have been validated in cell culture for targeting nucleases for genome editing and for targeting transcription activators (18,19). To test the ability of a TAL protein to serve as an affinity enrichment reagent for native chromatin isolation, a TAL protein was designed that bound a unique 18-nt region of DNA in the promoter region of the GAL1 gene in Saccharomyces cerevisiae (Figure 1B). We chose to analyze proteins and histone PTMs regulating the galactose-induced gene transcription of GAL1 because (1) this is a well-studied genomic locus, which will provide for proof-of-principle analysis, and (2) we previously used this locus to develop the ChAP-MS technique (13); thus, a comparison can be made to the TAL-ChAP-MS approach.Figure 1.


Purification of a specific native genomic locus for proteomic analysis.

Byrum SD, Taverna SD, Tackett AJ - Nucleic Acids Res. (2013)

TAL proteins can specifically enrich native chromatin sections. (A) Schematic overview of TAL-ChAP-MS technology. (B) A unique DNA sequence in the promoter region of GAL1 was used to design a specific binding TAL protein that contained a PrA affinity tag. (C) A pTAL-PrA plasmid was introduced into S. cerevisiae cells, and the constitutive expression of the TAL-PrA fusion protein was confirmed by western blotting for PrA. (D) Expression of TAL-PrA does not impede galactose-induced GAL1 transcription. cDNA from wild-type yeast and wild-type with a plasmid expressing PrA-tagged TAL (+pTAL-PrA) grown in glucose (Glu) or galactose (Gal) was used as a template for real time PCR analysis of GAL1 versus ACT1 gene transcription. Error bars are the standard deviation. (E) TAL-PrA specifically binds and enriches chromatin at the promoter of transcriptionally active GAL1. ChIP was performed to the PrA-tag in wild-types cells containing the TAL-PrA (+pTAL-PrA, light gray bars) and in wild-type control (dark gray bars). The efficiency of GAL1 enrichment relative to ACT1 was monitored by real-time PCR with primers targeted to the TAL binding site (‘0’) and to DNA sequences 2000 bp up- and downstream. The standard deviation is indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814360&req=5

gkt822-F1: TAL proteins can specifically enrich native chromatin sections. (A) Schematic overview of TAL-ChAP-MS technology. (B) A unique DNA sequence in the promoter region of GAL1 was used to design a specific binding TAL protein that contained a PrA affinity tag. (C) A pTAL-PrA plasmid was introduced into S. cerevisiae cells, and the constitutive expression of the TAL-PrA fusion protein was confirmed by western blotting for PrA. (D) Expression of TAL-PrA does not impede galactose-induced GAL1 transcription. cDNA from wild-type yeast and wild-type with a plasmid expressing PrA-tagged TAL (+pTAL-PrA) grown in glucose (Glu) or galactose (Gal) was used as a template for real time PCR analysis of GAL1 versus ACT1 gene transcription. Error bars are the standard deviation. (E) TAL-PrA specifically binds and enriches chromatin at the promoter of transcriptionally active GAL1. ChIP was performed to the PrA-tag in wild-types cells containing the TAL-PrA (+pTAL-PrA, light gray bars) and in wild-type control (dark gray bars). The efficiency of GAL1 enrichment relative to ACT1 was monitored by real-time PCR with primers targeted to the TAL binding site (‘0’) and to DNA sequences 2000 bp up- and downstream. The standard deviation is indicated.
Mentions: To alleviate genomic engineering for affinity enrichment of chromatin sections, we report the use of modified transcription activator-like (TAL) effector proteins to site-specifically target a native section of a chromosome for purification and proteomic analysis. We term this approach TAL-ChAP-MS (Figure 1A). TAL effector proteins are from Xanthomonas, which infects plants and translocates TAL effectors into cells where they serve as transcription activators (14–16). TALs contain a central domain of 18 tandem repeats of 34 amino acids each, which direct sequence-specific DNA binding (16,17). Binding to a given nucleobase in DNA is determined by two adjacent amino acids (12 and 13) within each of the 18 repeats (14). Thus, by mutating these amino acids in each of the 18 tandem repeats, one can ‘program’ binding to a given 18-nt region of DNA in vivo. TAL proteins have been validated in cell culture for targeting nucleases for genome editing and for targeting transcription activators (18,19). To test the ability of a TAL protein to serve as an affinity enrichment reagent for native chromatin isolation, a TAL protein was designed that bound a unique 18-nt region of DNA in the promoter region of the GAL1 gene in Saccharomyces cerevisiae (Figure 1B). We chose to analyze proteins and histone PTMs regulating the galactose-induced gene transcription of GAL1 because (1) this is a well-studied genomic locus, which will provide for proof-of-principle analysis, and (2) we previously used this locus to develop the ChAP-MS technique (13); thus, a comparison can be made to the TAL-ChAP-MS approach.Figure 1.

Bottom Line: Here, we describe an approach to isolate native chromatin sections without genomic engineering for label-free proteomic identification of associated proteins and histone post-translational modifications.The TAL-PrA fusion enabled chromatin affinity purification (ChAP) of a small section of native chromatin upstream from the GAL1 locus, permitting mass spectrometric (MS) identification of proteins and histone post-translational modifications regulating galactose-induced transcription.This TAL-ChAP-MS approach allows the biochemical isolation of a specific native genomic locus for proteomic studies and will provide for unprecedented objective insight into protein and epigenetic mechanisms regulating site-specific chromosome metabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA and Department of Pharmacology and Molecular Sciences, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.

ABSTRACT
Here, we describe an approach to isolate native chromatin sections without genomic engineering for label-free proteomic identification of associated proteins and histone post-translational modifications. A transcription activator-like (TAL) protein A fusion protein was designed to recognize a unique site in the yeast GAL1 promoter. The TAL-PrA fusion enabled chromatin affinity purification (ChAP) of a small section of native chromatin upstream from the GAL1 locus, permitting mass spectrometric (MS) identification of proteins and histone post-translational modifications regulating galactose-induced transcription. This TAL-ChAP-MS approach allows the biochemical isolation of a specific native genomic locus for proteomic studies and will provide for unprecedented objective insight into protein and epigenetic mechanisms regulating site-specific chromosome metabolism.

Show MeSH