A workflow for genome-wide mapping of archaeal transcription factors with ChIP-seq.
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.
University of California Davis, Department of Biomedical Engineering and Genome Center, One Shields Avenue, Davis, CA 95616, USA. egwilbanks@ucdavis.edu
Elizabeth G
EG
Wilbanks
David J
DJ
Larsen
Russell Y
RY
Neches
Andrew I
AI
Yao
Chia-Ying
CY
Wu
Rachel A S
RA
Kjolby
Marc T
MT
Facciotti
D019843:Archaeal Proteins / Q000378:metabolism*; D001665:Binding Sites; D047369:Chromatin Immunoprecipitation; D020745:Genome, Archaeal; D019617:Halobacterium salinarum / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D017422:Sequence Analysis, DNA; D012984:Software; D014157:Transcription Factors / Q000378:metabolism*
D019843:Archaeal Proteins; D014157:Transcription Factors