# Technologies for simultaneous characterization of regulatory activity and protein binding

> **NIH NIH R21** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2020 · $201,875

## Abstract

Project Summary
Mutations in gene regulatory elements are a major cause of human disease. Large-scale genomic assays, such
as ChIP-seq and ATAC-seq, have identified millions of putative regulatory elements across many different cell
types and tissues. Furthermore, massively parallel reporter assays (MPRAs), have allowed us to test thousands
of regulatory sequences and their variants for their functional activity in a high-throughput manner. In addition,
lentivirus-based MPRAs (lentiMPRAs) have enabled the testing of candidate sequences for regulatory activity
with high reproducibility in hard to transfect cells and in chromatin context via genomic integration. While these
assays have significantly expanded our knowledge of regulatory elements, technologies that can simultaneously
analyze both the regulatory function of a specific sequence and the transcription factors, cofactors and
epigenomic modifications that determine it do not exist. Here, we will develop a novel technology, crMPRA
(CUT&RUN MPRA), that combines two separate techniques, lentiMPRA and cleavage under targets and release
using nuclease (CUT&RUN) to simultaneously analyze in a high-throughput manner the regulatory activity,
protein binding and epigenetic modification of thousands of sequences. We will take advantage of lentiMPRA
both for testing thousands of candidate sequences for their regulatory activity, but also to enrich the genome with
thousands of integrations of a specific sequence, such that it could be assayed for protein binding and epigenetic
modifications via CUT&RUN. In Aim 1, we will develop crMPRA, by taking advantage of sequences that were
previously characterized via lentiMPRA (regulatory activity) and ChIP-seq (protein binding and epigenetic marks)
in hepatocellular carcinoma HepG2 cells. We will use these sequences to build an MPRA library and characterize
them for their regulatory activity via lentiMPRA. We will also simultaneously carry out CUT&RUN on specific TFs
(e.g. EP300, FOXA2, HNF4A) and epigenetic marks (e.g. H3K27ac, H3K27me3). For Aim 2, we will further test
a transcription factor binding site perturbation library via crMPRA how these perturbations affect regulatory
activity, protein binding and epigenetic modification, and analyze the functional correlation or independency
between these states. As such, this novel technology will allow us to increase our understanding of the regulatory
code at several levels including TF binding, histone modification, and transcriptional activation, and how its
alteration can lead to human disease.

## Key facts

- **NIH application ID:** 9998021
- **Project number:** 5R21HG010683-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** Nadav Ahituv
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $201,875
- **Award type:** 5
- **Project period:** 2019-08-16 → 2022-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9998021

## Citation

> US National Institutes of Health, RePORTER application 9998021, Technologies for simultaneous characterization of regulatory activity and protein binding (5R21HG010683-02). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/9998021. Licensed CC0.

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