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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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Distance from predicted TfbD-binding site for ChIP-seq (consensus between biological replicates), 500-bp tiling microarray ChIP-Chip (consensus between biological replicates) and 12-bp tiling microarray ChIP-Chip experiments. The observed difference in means was statistically significant (Mann Whitney U-test, p value < 0.005), as is the observed difference in variance (Bartlett test, p value < 0.005).
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gks063-F5: Distance from predicted TfbD-binding site for ChIP-seq (consensus between biological replicates), 500-bp tiling microarray ChIP-Chip (consensus between biological replicates) and 12-bp tiling microarray ChIP-Chip experiments. The observed difference in means was statistically significant (Mann Whitney U-test, p value < 0.005), as is the observed difference in variance (Bartlett test, p value < 0.005).

Mentions: ChIP enrichment of binding sites determined by qPCR and sequencing show a linear relationship. Data shown are drawn from multiple ChIP experiments: the Bat ChIP (Pbrp and PcrtB1 closed and open circles) and the TfbD ChIP and the reduced cell number TfbD ChIPs (PVNG906H and Patp_p; closed and open triangles). Differences in enrichment at the TfbD-bound promoters corresponded to changes produced by decreasing the number of cells in the ChIP reaction (see Figure 5 for further details).


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)

Distance from predicted TfbD-binding site for ChIP-seq (consensus between biological replicates), 500-bp tiling microarray ChIP-Chip (consensus between biological replicates) and 12-bp tiling microarray ChIP-Chip experiments. The observed difference in means was statistically significant (Mann Whitney U-test, p value < 0.005), as is the observed difference in variance (Bartlett test, p value < 0.005).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks063-F5: Distance from predicted TfbD-binding site for ChIP-seq (consensus between biological replicates), 500-bp tiling microarray ChIP-Chip (consensus between biological replicates) and 12-bp tiling microarray ChIP-Chip experiments. The observed difference in means was statistically significant (Mann Whitney U-test, p value < 0.005), as is the observed difference in variance (Bartlett test, p value < 0.005).
Mentions: ChIP enrichment of binding sites determined by qPCR and sequencing show a linear relationship. Data shown are drawn from multiple ChIP experiments: the Bat ChIP (Pbrp and PcrtB1 closed and open circles) and the TfbD ChIP and the reduced cell number TfbD ChIPs (PVNG906H and Patp_p; closed and open triangles). Differences in enrichment at the TfbD-bound promoters corresponded to changes produced by decreasing the number of cells in the ChIP reaction (see Figure 5 for further details).

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