# High-throughput functional characterization of human enhancers

> **NIH NIH UM1** · CORNELL UNIVERSITY · 2020 · $329,509

## Abstract

PROJECT SUMMARY/ABSTRACT
Specific enhancers interact with promoters to specify the cellular pattern, timing, and levels of gene expression.
Enhancers can reside up to megabases away from their target gene promoters and strongly activate
transcription. Aim 1 will characterize active enhancer elements and their relationship to promoter elements in
vivo in human K562 (a tier 1 ENCODE cell line) by testing a broad array of Transcription Regulatory Elements
(TREs) for their enhancer activity using eSTARR-seq, our modified element-clone-compatible STARR-seq
assay. This collection of TREs will be selected based on a variety of criteria established by ENCODE and others.
Large numbers of selected TREs can be handled using our new Clone- seq method, and then tested for enhancer
activity by eSTARR-seq. For the TREs that have significant enhancer activity, ~10,000 synthetic mutations will
be generated that are designed to destroy distinct TF binding motifs found within each enhancer. We will
generate mutant clones using our en masse Clone-seq2 method and examine their impact on enhancer activity
using eSTARR-seq. These data will be used to understand the underlying molecular architecture and function
of enhancers and promoters. Aim 2 generates K562 cell lines using CRISPR/Cas9 that contain critical synthetic
enhancer mutations identified in Aim 1. PRO- seq assays can then be used to measure with high sensitivity and
resolution the transcription at the variant enhancers as well as all TREs and transcription units genome-wide.
This will reveal the role of DNA sequence motifs within native enhancer loci in the regulatory crosstalk with distal
gene promoters and enhancers. Circularized Chromosome Conformation Capture (4C) experiments with
particular enhancers as the anchor site will provide an unbiased analysis of distal interactions, while targeted
ChIP-qPCR experiments will test effects of these mutant enhancers on transcription factor binding and local
histone marks at these genomic points of enhancer interaction. Thus, Aim 2 rigorously characterizes mutated
enhancers from Aim 1 in their native chromatin environment. Aim 3 characterizes the de novo activation of
enhancers, which are known to be triggered by the heat shock activation of HSF1, a master regulator. Because
the sequence motif, HSE, to which HSF1 binds is well defined, targeted HSE mutations that cripple the enhancer
activity will be made immediately using CRISPR at native loci and the effects on transcription genome-wide can
be analyzed directly by PRO-seq. Additional critical motifs in these inducible enhancers will be identified in a less
biased way by the more laborious, but high-throughput, eSTARR-seq approach described in Aim 1. Finally,
tracking the kinetics with which the structural characteristics of these enhancers form in the minutes following
heat shock relative to the induced transcriptional activity as measured by PRO-seq allows assessment of which
characteristics (DNase I hypersensit...

## Key facts

- **NIH application ID:** 10166068
- **Project number:** 3UM1HG009393-04S1
- **Recipient organization:** CORNELL UNIVERSITY
- **Principal Investigator:** JOHN T LIS
- **Activity code:** UM1 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $329,509
- **Award type:** 3
- **Project period:** 2020-09-25 → 2023-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10166068

## Citation

> US National Institutes of Health, RePORTER application 10166068, High-throughput functional characterization of human enhancers (3UM1HG009393-04S1). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10166068. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*