# HyCCAPP: A new method for the functional analysis of regulatory SNPs

> **NIH NIH R01** · WAKE FOREST UNIVERSITY HEALTH SCIENCES · 2021 · $487,175

## Abstract

Over the past decade, genome-wide association studies and comprehensive whole-genome sequencing
analyses have uncovered both common and rare variants that are associated with a wide range of disease-
related traits. The majority of these associated variants lie in intergenic regions, and it has been suggested that
they modulate the expression of individual genes. Indeed, genetic analyses have identified a large number of
expression quantitative trait loci adjacent to the affected genes (cis-eQTL), and many sequence variants are
strongly associated with both the expression of individual genes and disease-related traits. However,
deciphering the underlying molecular mechanisms has been challenging. Traditional laboratory approaches,
such as luciferase reporter constructs or electrophoretic mobility shift assays, as well as recent chromatin
analyses (DNase hypersensitivity mapping, ATAC-Seq) clearly suggest a functional impact for disease-
associated promoter variants, but no effective methods exist to identify the regulatory proteins binding to these
variant sites and mediating their effect on the regulation of gene expression. Therefore, alternative approaches
are required to more efficiently identify these unknown regulatory proteins whose promoter binding and
interaction is affected by eQTL variants.
 We recently developed a novel approach, Hybridization Capture of Chromatin-Associated Proteins for
Proteomics (HyCCAPP), that allows the identification of all proteins bound to a specific target chromatin region
by mass spectrometry. We propose to adapt the HyCCAPP approach for the analysis of luciferase reporter
plasmid constructs commonly used to assess the impact of sequence variants on promoter activity, and apply
the technology to the analysis of selected eQTL promoter variants. We hypothesize that the HyCCAPP
technology will uncover novel regulatory proteins mediating the effect of promoter variants on gene
expression, revealing potentially novel molecular mechanisms underlying eQTLs. We will pursue three
Specific Aims: 1) optimize HyCCAPP for the analysis of luciferase reporter plasmids, 2) analyze select
promoter variants in eQTL regions using plasmid HyCCAPP, and 3) validate the impact of protein binding on
gene expression in vivo in CRISPR-Cas9 edited cell lines.
 The technology development of HyCCAPP to target luciferase reporter plasmids, and a proof-of-principle
application to promoter variants associated with gene expression changes, will establish a powerful new and
effective tool for the investigation of the mechanisms by which regulatory sequence variants alter binding of
regulatory proteins. No other current technology allows the effective de novo identification of DNA binding
proteins affected by sequence variants. The use of commonly used luciferase reporter plasmid approaches
with the new HyCCAPP technology will help reveal new mechanisms of gene expression regulation
contributing to the development and progression of human dis...

## Key facts

- **NIH application ID:** 10245020
- **Project number:** 5R01GM118741-04
- **Recipient organization:** WAKE FOREST UNIVERSITY HEALTH SCIENCES
- **Principal Investigator:** MICHAEL OLIVIER
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $487,175
- **Award type:** 5
- **Project period:** 2018-09-18 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10245020

## Citation

> US National Institutes of Health, RePORTER application 10245020, HyCCAPP: A new method for the functional analysis of regulatory SNPs (5R01GM118741-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10245020. Licensed CC0.

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