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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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Spatial resolution of binding sites calculated from randomly subsampled Bat ChIP-seq datasets of decreasing coverage. The distance from ChIPseq predicted binding site to nearest Bat-binding motif was calculated for both the crtB1 (dark gray bars) and brp (light gray bars) promoter regions. Binding sites for in the crtB1 promoter could not be detected for datasets with 100 000 reads and below (no bars in plot).
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gks063-F8: Spatial resolution of binding sites calculated from randomly subsampled Bat ChIP-seq datasets of decreasing coverage. The distance from ChIPseq predicted binding site to nearest Bat-binding motif was calculated for both the crtB1 (dark gray bars) and brp (light gray bars) promoter regions. Binding sites for in the crtB1 promoter could not be detected for datasets with 100 000 reads and below (no bars in plot).

Mentions: For the Bat dataset, the more strongly enriched binding site at the brp promoter could be detected in datasets as small as 50 000 reads (0.64 x coverage), whereas the weaker binding at the crtB1 promoter was undetectable below 150 000 reads (1.9 x coverage). No false positives were detected in these lower coverage datasets, with the exception of a single site in the 200 000 read dataset. Examining the effect of decreased coverage on the spatial resolution, we found that the automated prediction of the binding site remained accurate, until just before the site became undetectable (Figure 8).Figure 8.


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)

Spatial resolution of binding sites calculated from randomly subsampled Bat ChIP-seq datasets of decreasing coverage. The distance from ChIPseq predicted binding site to nearest Bat-binding motif was calculated for both the crtB1 (dark gray bars) and brp (light gray bars) promoter regions. Binding sites for in the crtB1 promoter could not be detected for datasets with 100 000 reads and below (no bars in plot).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks063-F8: Spatial resolution of binding sites calculated from randomly subsampled Bat ChIP-seq datasets of decreasing coverage. The distance from ChIPseq predicted binding site to nearest Bat-binding motif was calculated for both the crtB1 (dark gray bars) and brp (light gray bars) promoter regions. Binding sites for in the crtB1 promoter could not be detected for datasets with 100 000 reads and below (no bars in plot).
Mentions: For the Bat dataset, the more strongly enriched binding site at the brp promoter could be detected in datasets as small as 50 000 reads (0.64 x coverage), whereas the weaker binding at the crtB1 promoter was undetectable below 150 000 reads (1.9 x coverage). No false positives were detected in these lower coverage datasets, with the exception of a single site in the 200 000 read dataset. Examining the effect of decreased coverage on the spatial resolution, we found that the automated prediction of the binding site remained accurate, until just before the site became undetectable (Figure 8).Figure 8.

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