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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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TfbD ChIP with decreasing numbers of cells. (A) ChIP-qPCR determined fold enrichment at two test promoters (VNG906H, dark bars and atp_p light bars) decreases with lower numbers of cells; however, strong (>5 x) enrichment is still observed with 3.5 × 108 cells. (B) For decreasing cell volume ChIP-seq experiments, fewer peaks could be identified (number of peaks identified, squares and lines), resulting in a significant loss in sensitivity. However, the percentage of identified peaks that were true positives stayed high (% true positives, triangles). True positives were defined as binding sites that were shared with at least one of the optimized 1.75 × 1010 ChIP TfbD biological replicates.
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gks063-F6: TfbD ChIP with decreasing numbers of cells. (A) ChIP-qPCR determined fold enrichment at two test promoters (VNG906H, dark bars and atp_p light bars) decreases with lower numbers of cells; however, strong (>5 x) enrichment is still observed with 3.5 × 108 cells. (B) For decreasing cell volume ChIP-seq experiments, fewer peaks could be identified (number of peaks identified, squares and lines), resulting in a significant loss in sensitivity. However, the percentage of identified peaks that were true positives stayed high (% true positives, triangles). True positives were defined as binding sites that were shared with at least one of the optimized 1.75 × 1010 ChIP TfbD biological replicates.

Mentions: We investigated the number of cells required for ChIP to produce sufficient enrichment at target protein-binding sites. Decreasing the number of cells in the ChIP reaction lowered the observed enrichment at target protein-binding sites; however, enrichment (>5 x) was detectable for highly enriched TfbD-binding sites when as few as 3.50 × 108 cells were used for ChIP, equivalent to 1 ml of a typical culture (Figure 6A). Because of the overall decrease in enrichment, smaller cell number ChIPs were less sensitive in binding site detection but maintained specificity (Figure 6B). The relatively small volume required for this ChIP assay should enable the high-throughput application of this method in the context of dynamic binding studies by allowing for the repetitive sampling of numerous strains with minimal perturbation.Figure 6.


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)

TfbD ChIP with decreasing numbers of cells. (A) ChIP-qPCR determined fold enrichment at two test promoters (VNG906H, dark bars and atp_p light bars) decreases with lower numbers of cells; however, strong (>5 x) enrichment is still observed with 3.5 × 108 cells. (B) For decreasing cell volume ChIP-seq experiments, fewer peaks could be identified (number of peaks identified, squares and lines), resulting in a significant loss in sensitivity. However, the percentage of identified peaks that were true positives stayed high (% true positives, triangles). True positives were defined as binding sites that were shared with at least one of the optimized 1.75 × 1010 ChIP TfbD biological replicates.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks063-F6: TfbD ChIP with decreasing numbers of cells. (A) ChIP-qPCR determined fold enrichment at two test promoters (VNG906H, dark bars and atp_p light bars) decreases with lower numbers of cells; however, strong (>5 x) enrichment is still observed with 3.5 × 108 cells. (B) For decreasing cell volume ChIP-seq experiments, fewer peaks could be identified (number of peaks identified, squares and lines), resulting in a significant loss in sensitivity. However, the percentage of identified peaks that were true positives stayed high (% true positives, triangles). True positives were defined as binding sites that were shared with at least one of the optimized 1.75 × 1010 ChIP TfbD biological replicates.
Mentions: We investigated the number of cells required for ChIP to produce sufficient enrichment at target protein-binding sites. Decreasing the number of cells in the ChIP reaction lowered the observed enrichment at target protein-binding sites; however, enrichment (>5 x) was detectable for highly enriched TfbD-binding sites when as few as 3.50 × 108 cells were used for ChIP, equivalent to 1 ml of a typical culture (Figure 6A). Because of the overall decrease in enrichment, smaller cell number ChIPs were less sensitive in binding site detection but maintained specificity (Figure 6B). The relatively small volume required for this ChIP assay should enable the high-throughput application of this method in the context of dynamic binding studies by allowing for the repetitive sampling of numerous strains with minimal perturbation.Figure 6.

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