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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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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).
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gks063-F4: 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).

Mentions: Independent biological replicates of the TfbD ChIP-seq experiment showed good reproducibility (82% overlapping binding sites). Differences between the two replicates are due to the smallest peaks in each dataset, as indicated by examining increasingly stringent enrichment thresholds (Supplementary Figure S5). Both binding sites in the Bat dataset and two example sites in the TfbD datasets (VNG906H and atp_p promoters) were confirmed with a ChIP-qPCR assay. The relationship between the quantification of ChIP enrichment at the binding sites determined by qPCR and sequencing was found to be well correlated across several experiments (Figure 4). The TfbD ChIP-seq binding sites agreed well with previously reported sites from TfbD ChIP-chip experiments (16). For both the ChIP-seq and ChIP-chip methods, 80% of consensus binding sites from biological replicates were identified by at least one of the replicates from the other method.Figure 4.


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)

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).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks063-F4: 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).
Mentions: Independent biological replicates of the TfbD ChIP-seq experiment showed good reproducibility (82% overlapping binding sites). Differences between the two replicates are due to the smallest peaks in each dataset, as indicated by examining increasingly stringent enrichment thresholds (Supplementary Figure S5). Both binding sites in the Bat dataset and two example sites in the TfbD datasets (VNG906H and atp_p promoters) were confirmed with a ChIP-qPCR assay. The relationship between the quantification of ChIP enrichment at the binding sites determined by qPCR and sequencing was found to be well correlated across several experiments (Figure 4). The TfbD ChIP-seq binding sites agreed well with previously reported sites from TfbD ChIP-chip experiments (16). For both the ChIP-seq and ChIP-chip methods, 80% of consensus binding sites from biological replicates were identified by at least one of the replicates from the other method.Figure 4.

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