# Functional Mapping of Enhancer Conservation Between Species to Enable Mechanistic Insights into Polygenic Disease

> **NIH NIH R35** · JACKSON LABORATORY · 2022 · $519,158

## Abstract

PROJECT SUMMARY
Recent advances to characterize cis-regulatory elements (CRE), including massively parallel reporter assays
and CRISPR-based screens of non-coding elements, have transformed our ability to comprehensively
characterize the non-coding genome at scale. Large scale efforts by us and others through the Encyclopedia of
DNA Elements (ENCODE) consortium are now underway to apply these methods genome-wide across many
cellular states. The results of these screens will have a transformative impact on our ability to read and write
the regulatory grammar of the cell. One direct application will be in the interpretation of causal alleles for
human disease risk and other phenotypic traits identified through genome-wide association studies. From
these studies we now know the majority of heritability for complex traits resides in non-coding regions of the
genome. Until recently it has been difficult to pinpoint individual causal alleles but progress is now being made
to identify and elucidate their molecular function. Despite our burgeoning success in understanding how a
variant impacts molecular phenotypes (e.g. gene transcription), we lack the ability to systematically evaluate
allele(s) within model organisms to understand their impact on physiological function. This disconnect is
partially due to our inability to identify the homologous non-coding region to target within model organisms. To
aid in modeling human regulatory variation in the mouse, in this project we will develop improved maps of
homologous CREs between human and mouse. Current comparative approaches rely on sequence homology
and correlative measures of gene expression such as regions of DNase hypersensitivity and chromatin
modifications. While these methods have provided valuable insight, they lack direct quantitative measurements
of a CRE's impact on individual genes and the location of the cis-regulatory modules (CRMs) within the CREs
responsible for activity. To overcome these shortcomings, in this study we will develop maps of CRE
conservation based directly on function. To accomplish this, we will differentiate induced pluripotent stem cells
(iPSCs) from human and mouse to early developmental states as the starting material for screens of CRE
activity. We will use (i) a CRISPR-based screen to endogenously perturb putative CREs important for neuronal
and epithelial function; and (ii) CREs with concordant and discordant activity across the two species will then
undergo saturation mutagenesis using a massively parallel reporter assay (MPRA). Results from the MPRA
will identify CRMs (e.g. TF binding motifs) within each CRE driving regulatory activity of the element. We will
use the results from both screens to construct improved maps of CRE conservation that will inform how to copy
the effects of genetic variation residing at these regions across species. Doing so will accelerate our progress
in moving human disease variants into animal models, thereby allowing us to better un...

## Key facts

- **NIH application ID:** 10491357
- **Project number:** 5R35HG011329-02
- **Recipient organization:** JACKSON LABORATORY
- **Principal Investigator:** Ryan Tewhey
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $519,158
- **Award type:** 5
- **Project period:** 2021-09-20 → 2026-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10491357

## Citation

> US National Institutes of Health, RePORTER application 10491357, Functional Mapping of Enhancer Conservation Between Species to Enable Mechanistic Insights into Polygenic Disease (5R35HG011329-02). Retrieved via AI Analytics 2026-06-11 from https://api.ai-analytics.org/grant/nih/10491357. Licensed CC0.

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