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A workflow for genome-wide mapping of archaeal transcription factors with ChIP-seq.

Wilbanks EG, Larsen DJ, Neches RY, Yao AI, Wu CY, Kjolby RA, Facciotti MT - Nucleic Acids Res. (2012)

Bottom Line: Chromosomal tagging of target proteins with a compact epitope facilitates a standardized and cost-effective workflow that is compatible with high-throughput immunoprecipitation of natively expressed transcription factors.While this study focuses on the application of ChIP-seq in H. salinarum sp.NRC-1, our workflow can also be adapted for use in other archaea and bacteria with basic genetic tools.

View Article: PubMed Central - PubMed

Affiliation: University of California Davis, Department of Biomedical Engineering and Genome Center, One Shields Avenue, Davis, CA 95616, USA. egwilbanks@ucdavis.edu

ABSTRACT
Deciphering the structure of gene regulatory networks across the tree of life remains one of the major challenges in postgenomic biology. We present a novel ChIP-seq workflow for the archaea using the model organism Halobacterium salinarum sp. NRC-1 and demonstrate its application for mapping the genome-wide binding sites of natively expressed transcription factors. This end-to-end pipeline is the first protocol for ChIP-seq in archaea, with methods and tools for each stage from gene tagging to data analysis and biological discovery. Genome-wide binding sites for transcription factors with many binding sites (TfbD) are identified with sensitivity, while retaining specificity in the identification the smaller regulons (bacteriorhodopsin-activator protein). Chromosomal tagging of target proteins with a compact epitope facilitates a standardized and cost-effective workflow that is compatible with high-throughput immunoprecipitation of natively expressed transcription factors. The Pique package, an open-source bioinformatics method, is presented for identification of binding events. Relative to ChIP-Chip and qPCR, this workflow offers a robust catalog of protein-DNA binding events with improved spatial resolution and significantly decreased cost. While this study focuses on the application of ChIP-seq in H. salinarum sp. NRC-1, our workflow can also be adapted for use in other archaea and bacteria with basic genetic tools.

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Epitope tag-in approach for Hb. NRC-1. The Hb. NRC-1 ΔpyrF strain is transformed with a plasmid containing the mevinolin-resistance determinant (MevR; dark gray box) and the pyrF gene (black box) that confers 5-FOA sensitivity. The plasmid carries an engineered sequence containing the HA epitope sequence (white box) flanked by the last 500 bp of the target gene and the 500 bp downstream of the target gene (light gray boxes). Plasmid sequence is shown as solid line, chromosomal sequence is shown as solid, wavy lines. Cross-over can occur between target gene (light gray box) and flanking sequence (gray wavy line) in the chromosome and the homologous regions in the plasmid sequence, at either position 1 or 2 (position 1 example shown). PCR screening of mevinolin-resistant colonies is used to determine successful first recombinants. Subsequent plating on 5-FOA selects for second recombinants (via counter-selection with the pyrF gene). In this example, a second cross-over at site 2 produces the desired chromosomally integrated recombinant target_gene::HA fusion. PCR screening of these colonies is required to distinguish this desired second recombinant from a second recombinant occurring at position 1. Drawing is not to scale.
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gks063-F1: Epitope tag-in approach for Hb. NRC-1. The Hb. NRC-1 ΔpyrF strain is transformed with a plasmid containing the mevinolin-resistance determinant (MevR; dark gray box) and the pyrF gene (black box) that confers 5-FOA sensitivity. The plasmid carries an engineered sequence containing the HA epitope sequence (white box) flanked by the last 500 bp of the target gene and the 500 bp downstream of the target gene (light gray boxes). Plasmid sequence is shown as solid line, chromosomal sequence is shown as solid, wavy lines. Cross-over can occur between target gene (light gray box) and flanking sequence (gray wavy line) in the chromosome and the homologous regions in the plasmid sequence, at either position 1 or 2 (position 1 example shown). PCR screening of mevinolin-resistant colonies is used to determine successful first recombinants. Subsequent plating on 5-FOA selects for second recombinants (via counter-selection with the pyrF gene). In this example, a second cross-over at site 2 produces the desired chromosomally integrated recombinant target_gene::HA fusion. PCR screening of these colonies is required to distinguish this desired second recombinant from a second recombinant occurring at position 1. Drawing is not to scale.

Mentions: Hb. NRC-1 ΔpyrF was transformed with a recombinant plasmid that contained the terminal 500 bp of the target gene, a hemagglutinin (HA) tag, stop codon and 500 bp downstream sequence (Figure 1). Homologous recombination between the chromosomal target gene and the recombinant plasmid sequence introduced the HA tag to the chromosomal sequence. Successful first recombinants were determined by PCR screening of MevR colonies (Supplementary Figure S1). The plasmid was subsequently resolved using 5-FOA counter-selection as previously described (22,30). Strains with C-terminally HA-tagged target proteins were further verified by PCR and Sanger sequencing (Supplementary Figures S1–S2 and Supplementary Information). For the rapid construction of epitope-tagged transcription factor strains, this general strategy of utilizing DNA synthesis and homologous recombination-based chromosomal modification can be readily extended to any organisms with a system for targeted genetic knockouts.Figure 1.


A workflow for genome-wide mapping of archaeal transcription factors with ChIP-seq.

Wilbanks EG, Larsen DJ, Neches RY, Yao AI, Wu CY, Kjolby RA, Facciotti MT - Nucleic Acids Res. (2012)

Epitope tag-in approach for Hb. NRC-1. The Hb. NRC-1 ΔpyrF strain is transformed with a plasmid containing the mevinolin-resistance determinant (MevR; dark gray box) and the pyrF gene (black box) that confers 5-FOA sensitivity. The plasmid carries an engineered sequence containing the HA epitope sequence (white box) flanked by the last 500 bp of the target gene and the 500 bp downstream of the target gene (light gray boxes). Plasmid sequence is shown as solid line, chromosomal sequence is shown as solid, wavy lines. Cross-over can occur between target gene (light gray box) and flanking sequence (gray wavy line) in the chromosome and the homologous regions in the plasmid sequence, at either position 1 or 2 (position 1 example shown). PCR screening of mevinolin-resistant colonies is used to determine successful first recombinants. Subsequent plating on 5-FOA selects for second recombinants (via counter-selection with the pyrF gene). In this example, a second cross-over at site 2 produces the desired chromosomally integrated recombinant target_gene::HA fusion. PCR screening of these colonies is required to distinguish this desired second recombinant from a second recombinant occurring at position 1. Drawing is not to scale.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3378898&req=5

gks063-F1: Epitope tag-in approach for Hb. NRC-1. The Hb. NRC-1 ΔpyrF strain is transformed with a plasmid containing the mevinolin-resistance determinant (MevR; dark gray box) and the pyrF gene (black box) that confers 5-FOA sensitivity. The plasmid carries an engineered sequence containing the HA epitope sequence (white box) flanked by the last 500 bp of the target gene and the 500 bp downstream of the target gene (light gray boxes). Plasmid sequence is shown as solid line, chromosomal sequence is shown as solid, wavy lines. Cross-over can occur between target gene (light gray box) and flanking sequence (gray wavy line) in the chromosome and the homologous regions in the plasmid sequence, at either position 1 or 2 (position 1 example shown). PCR screening of mevinolin-resistant colonies is used to determine successful first recombinants. Subsequent plating on 5-FOA selects for second recombinants (via counter-selection with the pyrF gene). In this example, a second cross-over at site 2 produces the desired chromosomally integrated recombinant target_gene::HA fusion. PCR screening of these colonies is required to distinguish this desired second recombinant from a second recombinant occurring at position 1. Drawing is not to scale.
Mentions: Hb. NRC-1 ΔpyrF was transformed with a recombinant plasmid that contained the terminal 500 bp of the target gene, a hemagglutinin (HA) tag, stop codon and 500 bp downstream sequence (Figure 1). Homologous recombination between the chromosomal target gene and the recombinant plasmid sequence introduced the HA tag to the chromosomal sequence. Successful first recombinants were determined by PCR screening of MevR colonies (Supplementary Figure S1). The plasmid was subsequently resolved using 5-FOA counter-selection as previously described (22,30). Strains with C-terminally HA-tagged target proteins were further verified by PCR and Sanger sequencing (Supplementary Figures S1–S2 and Supplementary Information). For the rapid construction of epitope-tagged transcription factor strains, this general strategy of utilizing DNA synthesis and homologous recombination-based chromosomal modification can be readily extended to any organisms with a system for targeted genetic knockouts.Figure 1.

Bottom Line: Chromosomal tagging of target proteins with a compact epitope facilitates a standardized and cost-effective workflow that is compatible with high-throughput immunoprecipitation of natively expressed transcription factors.While this study focuses on the application of ChIP-seq in H. salinarum sp.NRC-1, our workflow can also be adapted for use in other archaea and bacteria with basic genetic tools.

View Article: PubMed Central - PubMed

Affiliation: University of California Davis, Department of Biomedical Engineering and Genome Center, One Shields Avenue, Davis, CA 95616, USA. egwilbanks@ucdavis.edu

ABSTRACT
Deciphering the structure of gene regulatory networks across the tree of life remains one of the major challenges in postgenomic biology. We present a novel ChIP-seq workflow for the archaea using the model organism Halobacterium salinarum sp. NRC-1 and demonstrate its application for mapping the genome-wide binding sites of natively expressed transcription factors. This end-to-end pipeline is the first protocol for ChIP-seq in archaea, with methods and tools for each stage from gene tagging to data analysis and biological discovery. Genome-wide binding sites for transcription factors with many binding sites (TfbD) are identified with sensitivity, while retaining specificity in the identification the smaller regulons (bacteriorhodopsin-activator protein). Chromosomal tagging of target proteins with a compact epitope facilitates a standardized and cost-effective workflow that is compatible with high-throughput immunoprecipitation of natively expressed transcription factors. The Pique package, an open-source bioinformatics method, is presented for identification of binding events. Relative to ChIP-Chip and qPCR, this workflow offers a robust catalog of protein-DNA binding events with improved spatial resolution and significantly decreased cost. While this study focuses on the application of ChIP-seq in H. salinarum sp. NRC-1, our workflow can also be adapted for use in other archaea and bacteria with basic genetic tools.

Show MeSH
Related in: MedlinePlus