# Understanding transcriptional silencing & anti-silencing mechanisms in Shigella

> **NIH NIH R15** · UNIVERSITY OF NEVADA LAS VEGAS · 2020 · $444,540

## Abstract

Summary
Nucleoid-associated proteins (NAPs), like the histone-like nucleoid structuring protein (H-NS), bind to DNA and
transcriptionally silence genes in bacteria. DNA-binding proteins known as anti-silencers, function to offset
NAP-mediated silencing, leading to gene expression. These frequent and widespread regulatory events in
bacteria are functionally akin to eukaryotic chromatin remodeling and control many aspects of bacterial
physiology, including virulence. Since bacterial NAPs (“the silencers”) are crucial for the organization and
sequestration of bacterial DNA, insight into these key regulatory processes will likely lead to the discovery of
novel drug targets and new therapeutics. While many unrelated DNA-binding proteins can serve as anti-
silencers, mechanistic underpinnings are likely shared. Consequently, the long-term goal of this project is to
fully understand transcriptional silencing and anti-silencing mechanisms in the bacterial pathogen Shigella. In
Shigella, at 30°C or lower, H-NS silences many genes on the large virulence plasmid (pINV; 230 kb). Upon a
switch to human body temperature (37°C), production of the VirB anti-silencer counters H-NS silencing leading
to the up-regulation of key virulence genes. Research findings made through previous R15 awards are
foundational to the work currently proposed. The overarching hypothesis of this study is “VirB spreading
and/or bridging activities on DNA directly relieve H-NS mediated silencing by triggering a loss of negative
supercoils in DNA.” This hypothesis will be tested using two specific aims. In Aim 1, the relationship between
DNA supercoiling and transcriptional silencing/anti-silencing will be determined using in vivo and in vitro
approaches. First, two creative and complementary in vitro approaches that ‘fix’ VirB-mediated changes in
plasmid DNA will be used to determine if VirB:DNA interactions directly trigger a change in DNA supercoiling.
Second, the effect of DNA supercoiling on H-NS-mediated silencing is investigated by measuring the activity of
an H-NS-silenced promoter in either a set of topoisomerase I mutants or in the presence of the DNA gyrase
inhibitor, novobiocin. In Aim 2, the nature of VirB:DNA complexes and their involvement in VirB-dependent
transcriptional anti-silencing will be elucidated. First, ChIP-PCR combined with an innovative DNase I
treatment step will definitively test if VirB spreads along DNA from its cognate site at the well-characterized
icsP locus. Second, VirB mutants likely defective in VirB:VirB interactions will be characterized in terms of
binding, spreading, forming foci in the bacterial cytoplasm and anti-silencing activities. Third, the involvement
of VirB spreading and/or bridging in the anti-silencing of the icsP promoter is investigated. The work proposed
is in line with the stated long-term goal (above) and is likely to provide insight into transcriptional silencing/anti-
silencing mechanisms in other bacteria. All experiments a...

## Key facts

- **NIH application ID:** 10045805
- **Project number:** 2R15AI090573-04
- **Recipient organization:** UNIVERSITY OF NEVADA LAS VEGAS
- **Principal Investigator:** HELEN J WING
- **Activity code:** R15 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $444,540
- **Award type:** 2
- **Project period:** 2010-05-01 → 2023-12-03

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10045805

## Citation

> US National Institutes of Health, RePORTER application 10045805, Understanding transcriptional silencing & anti-silencing mechanisms in Shigella (2R15AI090573-04). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10045805. Licensed CC0.

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