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Profiling large-scale protein occupancy on bacterial genomes using IPOD-HR.

Rebecca L Hurto¹, Jeremy W Schroeder¹, Julian Trouillon²

¹Department of Biological Chemistry, University of Michigan, Ann Arbor, MI, USA.

Nature Protocols

|April 28, 2026

Summary

This summary is machine-generated.

in vivo protein occupancy display-high resolution (IPOD-HR) enables genome-wide profiling of protein-bound DNA in bacteria. This method overcomes limitations of other techniques, offering a more accessible way to study bacterial gene regulation.

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Heuristic Mining of Hierarchical Genotypes and Accessory Genome Loci in Bacterial Populations

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Area of Science:

Microbiology
Genomics
Molecular Biology

Background:

Understanding bacterial gene regulation requires identifying genomic regions bound by proteins.
Existing methods like ChIP-seq and ChIP-exo are labor-intensive and costly for comprehensive analysis.
ATAC-seq is unsuitable for bacteria due to interference from nucleoid-associated proteins.

Purpose of the Study:

To introduce in vivo protein occupancy display-high resolution (IPOD-HR) for genome-wide protein occupancy profiling in prokaryotes.
To provide a method for identifying condition- and genotype-dependent changes in protein occupancy linked to gene regulation.

Main Methods:

Developed and described the IPOD-HR protocol for profiling protein-bound DNA in bacterial systems.
Coupled IPOD-HR with RNA polymerase ChIP and subsequent high-throughput sequencing.
Utilized open-source, automated software for downstream data analysis.

Main Results:

IPOD-HR successfully profiles protein-bound DNA across bacterial genomes.
The method is independent of the specific protein identity.
Identified condition- and genotype-specific changes in protein occupancy.

Conclusions:

IPOD-HR is a powerful and accessible method for studying bacterial gene regulation.
It overcomes limitations of previous techniques, enabling detailed analysis of protein-DNA interactions.
The protocol includes a user-friendly computational pipeline for data analysis.