# Bacteriophage Mu as Tool to Study Genome Organization in Bacteria and Eukaryotes

> **NIH NIH R21** · UNIVERSITY OF TEXAS AT AUSTIN · 2021 · $447,069

## Abstract

Bacteriophage Mu as Tool to Study Genome Organization in Bacteria and Eukaryotes
The 3D configuration of the genome is complex, dynamic and crucial for gene regulation. The majority of
recent insights into genome conformations have been made using proximity-ligation based chromosome
conformation capture methods (e.g., 3C and HiC) and fluorescent in situ hybridization (FISH) techniques.
Drawbacks of both approaches are that they require chemical fixation, and in many cases require specification
of a small number of target sites on the genome to be tracked. Proximity ligation approaches selectively probe
only DNA-protein mediated interactions, have different efficiencies of detecting contacts with varying spatial
distances either within the same chromosome or between different chromosomes, and require several
additional in vitro steps after chemical crosslinking for obtaining and processing the data. We have developed
a new methodology that requires no chemical fixation or external perturbation and monitors DNA-DNA contact
frequencies in live cells. The methodology exploits the transposition mechanism of bacteriophage Mu, and has
been applied successfully to interrogate the 3D conformation of the E. coli genome. In contrast to the
dominance of short-range contacts seen with 3C/HiC, the Mu methodology captured all genomic contacts,
revealing that the genome was well-mixed. The methodology revealed widespread clustering of genetic loci in
3D space, many of the clusters consisting of co-regulated genes, which we subsequently validated using
fluorescence-based measurements. These key features of the E. coli genome – generalized mixing with
specific robust long-range contacts -- has not been detectable in studies using proximity ligation based
methods. Our measurements using Mu also revealed that proteins that compact DNA (condensin and a
histone-like protein) are responsible for the extensive long-range genomic contacts. In short, our Mu-based
measurements changed the static, short-range contact view of the E. coli genome generated by 3C/HiC
techniques to that of a dynamic chromosome anchored by specific contacts between biologically important
regions. We propose to extend the Mu methodology to bacteria that are not a natural host for Mu in order to
assess the universality of our findings among Bacteria, as well as to eukaryotes (first yeast and eventually
mammalian cell lines) by designing Mu vectors that will function in cells of each target species. Given that
chromatin folding is a major feature of gene regulation, and changes dynamically in development and disease,
it is imperative that we assess genome architecture in live cells. Our Mu transposition based methods provide
a new opportunity to unveil chromosome conformations without relying on the assumptions of proximity ligation
experiments. Our ability to accurately track chromosomal conformations will open new avenues for disease
diagnostics, disease target discovery and identification of stru...

## Key facts

- **NIH application ID:** 10265837
- **Project number:** 1R21HG011838-01
- **Recipient organization:** UNIVERSITY OF TEXAS AT AUSTIN
- **Principal Investigator:** Lydia Petra Freddolino
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $447,069
- **Award type:** 1
- **Project period:** 2021-09-22 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10265837

## Citation

> US National Institutes of Health, RePORTER application 10265837, Bacteriophage Mu as Tool to Study Genome Organization in Bacteria and Eukaryotes (1R21HG011838-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10265837. Licensed CC0.

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