# High-throughput functional characterization of human enhancers

> **NIH NIH UM1** · CORNELL UNIVERSITY · 2021 · $750,521

## Abstract

PROJECT SUMMARY/ABSTRACT:
The genomes of higher organisms and their expression are highly regulated in response to a variety of
developmental, environmental, and nutritional cues. The failure to execute proper gene regulation can lead to
developmental defects and disease states. The overarching goal of this research is to understand basic
regulatory mechanisms of genes at the level of transcription, the stage where RNA polymerase II (Pol II)
transcribes the genes into mRNA. This regulation is conducted by the concerted effort of DNA elements
(promoters and enhancers) and many protein machineries such as Pol II, TFs, and chromatin/histone modifying
complexes. Recent studies in Drosophila and mammalian cells have revealed that transcription is primarily
regulated at two rate-limiting steps; 1) Recruitment of Pol II to promoter and Pol II pausing at 20-50bp
downstream of transcription initiation site, and 2) Release of paused Pol II to productive elongation. A battery of
complementary approaches (some novel) are designed to identify human TFs and chromatin remodelers that act
at these steps and then rigorously address the mechanism by which they exert transcriptional regulation. by TFs
and chromatin modifiers with the highest possible spatiotemporal resolution genome-wide in distinct cell lines,
including K562, HCT116, HEK293, and undifferentiated and differentiated WTC-11. The wealth of existing
information in these cell lines will be complemented with Run-On assays (RO-seq), and others when necessary,
to identify TFs that act primarily on one or both of the rate-limiting regulated steps in transcription. These
observational assays include multiple highly-sensitive RO-seq assays for mapping the position and amount of
transcription across genes and enhancers genome-wide, ChIP-exo for detecting TF binding and occupancy,
STARR-seq assays to measure transcription regulatory activity of enhancers and promoters, and a degron
system to rapidly degrade TFs to assess their primary effects by these ‘-seq’ assays. Together, these systems
will test our regulatory models and assess their generality. In Aim 1, using a novel aptamer-based pulldown and
mass spectrometry approach, we will identify pioneering TFs that work through remodeling complexes such as
PBAP (SWI/SNF) to open chromatin for subsequent TF and Pol II recruitment. In Aim 2, we seek to identify cell-
type-specific TFs that act at major regulated steps of transcription, Steps 1, 2, or both. This will be achieved by a
comparative analysis of cell line specific nascent transcription profiles. Aim 3 will test the role of TF candidates,
which are associated with one or both of the regulatory steps in the transcription cycle, in living cells using a
Degron approach and multiple ‘-seq’ assays. In Aim 4, we will assess the roles of TFs in the interplay of
enhancers and promoters interactions using high-throughput episomal and chromosomal enhancer assays. The
proposal will identify TFs that act at specific ...

## Key facts

- **NIH application ID:** 10241101
- **Project number:** 3UM1HG009393-04S2
- **Recipient organization:** CORNELL UNIVERSITY
- **Principal Investigator:** JOHN T LIS
- **Activity code:** UM1 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $750,521
- **Award type:** 3
- **Project period:** 2020-08-18 → 2023-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10241101

## Citation

> US National Institutes of Health, RePORTER application 10241101, High-throughput functional characterization of human enhancers (3UM1HG009393-04S2). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10241101. Licensed CC0.

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