# High-throughput inverted reporter assay for characterization of silencers and enhancer blockers

> **NIH NIH R21** · UNIVERSITY OF MICHIGAN AT ANN ARBOR · 2024 · $166,172

## Abstract

Project Summary
Cis-regulatory elements (CREs) have been shown to play a significant role in proper gene expression programs
and perturbations of these regions contribute to the development and progression of cancer. While many
examples of the consequences of misregulated positive regulatory elements, including enhancers and
promoters, exist, comparatively little is known about the role of disrupted negative regulatory elements (NREs)
such as silencers and enhancer blockers. Recent evidence suggests that silencers are as abundant as
enhancers and enriched for disease associated variants, indicating this imbalance is not due to genomic NRE
frequency. Rather, NREs are understudied due to lack of a high-throughput reporter assay similar to those that
accelerated enhancer identification. Currently the only standard for locating NREs in the genome involves assays
that rely on a reduction or loss of reporter signal. This results in high false positive rates due to the multiple
artefacts inherent to pooled high-throughput assays which can cause reduction in signal. High noise and low
signal from RNA-seq pools, where strong repressive elements are represented by the fewest transcripts, produce
high false negative rates. This vastly increases the sequencing depth needed to accurately detect low transcript
levels of potent NREs. As a result of these challenges, replicability of NRE assay results have been poor. We
propose to address this gap by developing a high-throughput reporter assay system where strong NREs
generate a positive signal, observed as an increase in reporter gene transcription, through the use of a dCas9-
sgRNA system. Our dCas9 Inverted Reporter Assay (ht-dCIRA) overcomes limitations of traditional NRE reporter
assays with the use of signal inversion, allowing for robust and reliable identification of silencers and enhancer
blockers in the human genome. Improving replicability will allow the detection of NREs that are differentially
active across multiple samples, making comparisons of the regulatory elements driving normal vs tumor states
or response to drug treatment feasible. Reduction of sequencing depth requirements and reliable results with
fewer replicates will additionally make this technology applicable for use across a broad set of cancer cell lines.
This will expedite identification of novel silencers and enhancer blockers that are common to cancer states versus
NREs that vary among cell types. We will demonstrate the applicability of our assay in a genome-wide screen
for silencers and enhancer blockers in MCF-7 breast cancer cells. While many studies have examined the
response of MCF-7 cells to 17β-estradiol and subsequent activation of enhancers by estrogen receptor one
(ERα), little is known about the ERα-NRE relationship. We will apply ht-dCIRA to identify active NREs in both
17β-estradiol treated and untreated MCF-7 cells to determine for the first time silencers and enhancer blockers
that are differentially activated in t...

## Key facts

- **NIH application ID:** 10771001
- **Project number:** 5R21CA257864-03
- **Recipient organization:** UNIVERSITY OF MICHIGAN AT ANN ARBOR
- **Principal Investigator:** Alan P Boyle
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $166,172
- **Award type:** 5
- **Project period:** 2022-02-23 → 2025-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10771001

## Citation

> US National Institutes of Health, RePORTER application 10771001, High-throughput inverted reporter assay for characterization of silencers and enhancer blockers (5R21CA257864-03). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10771001. Licensed CC0.

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