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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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Related in: MedlinePlus

The 5′ to 3′ sequencing requirement and short read length produce stranded bias in sequence coverage. The shaded blue oval represents the protein of interest bound to DNA (solid black lines). Wavy lines represent either sense (blue) or antisense (red) DNA fragments from ChIP enrichment. The thicker portion of the line indicates regions sequenced by short read sequencing technologies. Sequenced tags are aligned to a reference genome and shown below is the strand-specific sequence coverage at each position in the genome. Punctate-binding events (e.g. transcription factors) are characterized by well-defined strand-specific bimodality in sequence coverage.
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gks063-F2: The 5′ to 3′ sequencing requirement and short read length produce stranded bias in sequence coverage. The shaded blue oval represents the protein of interest bound to DNA (solid black lines). Wavy lines represent either sense (blue) or antisense (red) DNA fragments from ChIP enrichment. The thicker portion of the line indicates regions sequenced by short read sequencing technologies. Sequenced tags are aligned to a reference genome and shown below is the strand-specific sequence coverage at each position in the genome. Punctate-binding events (e.g. transcription factors) are characterized by well-defined strand-specific bimodality in sequence coverage.

Mentions: These punctate target protein DNA-binding events produce a distinctive bimodal, strand-specific enrichment pattern in sequence coverage (Figure 2). This enrichment pattern was leveraged to identify binding sites using our open source software package Pique, which reports the candidate binding site’s enrichment as the ratio of sequence coverage in the IP data relative to a background control (Neches,R.Y., Wilbanks,E.G. and Facciotti,M.T., in preparation). Pique is implemented from a user-friendly graphical user interface and exports predicted binding sites to the Gaggle Genome Browser (18). From the Gaggle genome browser, users can explore and curate the data before proceeding with analysis via other downstream Gaggle tools (Figure 3).Figure 2.


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)

The 5′ to 3′ sequencing requirement and short read length produce stranded bias in sequence coverage. The shaded blue oval represents the protein of interest bound to DNA (solid black lines). Wavy lines represent either sense (blue) or antisense (red) DNA fragments from ChIP enrichment. The thicker portion of the line indicates regions sequenced by short read sequencing technologies. Sequenced tags are aligned to a reference genome and shown below is the strand-specific sequence coverage at each position in the genome. Punctate-binding events (e.g. transcription factors) are characterized by well-defined strand-specific bimodality in sequence coverage.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks063-F2: The 5′ to 3′ sequencing requirement and short read length produce stranded bias in sequence coverage. The shaded blue oval represents the protein of interest bound to DNA (solid black lines). Wavy lines represent either sense (blue) or antisense (red) DNA fragments from ChIP enrichment. The thicker portion of the line indicates regions sequenced by short read sequencing technologies. Sequenced tags are aligned to a reference genome and shown below is the strand-specific sequence coverage at each position in the genome. Punctate-binding events (e.g. transcription factors) are characterized by well-defined strand-specific bimodality in sequence coverage.
Mentions: These punctate target protein DNA-binding events produce a distinctive bimodal, strand-specific enrichment pattern in sequence coverage (Figure 2). This enrichment pattern was leveraged to identify binding sites using our open source software package Pique, which reports the candidate binding site’s enrichment as the ratio of sequence coverage in the IP data relative to a background control (Neches,R.Y., Wilbanks,E.G. and Facciotti,M.T., in preparation). Pique is implemented from a user-friendly graphical user interface and exports predicted binding sites to the Gaggle Genome Browser (18). From the Gaggle genome browser, users can explore and curate the data before proceeding with analysis via other downstream Gaggle tools (Figure 3).Figure 2.

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