# Functional Architecture and Interplay of Transcription Regulatory Elements of the Human Genome

> **NIH NIH R01** · CORNELL UNIVERSITY · 2024 · $680,349

## Abstract

PROJECT SUMMARY/ABSTRACT
Human genome is peppered with an estimated one million enhancers that can regulate their specific target
gene(s) from a distance up to megabases away, and in an orientation-independent manner. The broad goal of
this project is to define the fundamental architecture and function of human enhancers in a in-depth manner
and to better understand the specificity of their interplay with different classes of promoters that use different
transcription factors and/or are regulated at distinct steps in transcription. We have shown that enhancers can
be identified and precisely mapped using our GRO/PRO-cap assay that map transcription start sites of nascent
RNA with highest sensitivity of all available methods. This assay has shown that both enhancers and
promoters share a common architecture whereby both are delimited by two divergent core promoters (CPs)
and a central cluster of transcription factor (TF) binding motifs. The roles and required organization of the
multiple sequence motifs that constitute enhancers and the two CPs need to be fully dissected to understand
how active enhancers function. Additionally, enhancers can interact productively with specific promoters, and
the basis of this specificity especially at long range remains ill-defined. Finally, we know that promoter and
enhancer elements have sequence motifs that can act at distinct regulatory steps of the transcription cycle, but
how these activities coordinate gene regulation has yet to be examined. In Aim 1, we will test the activity of all
PRO-cap identified enhancer candidates in K562 from representative human Chromosomes 8 and 11 with a
carefully chosen set of promoters harboring distinct regulatory features. A set of active enhancer-promoter
combinations will then be subjected to a comprehensive motif mutagenesis of the central clusters of TF binding
motifs and each of the two core promoters. These studies test our fundamental enhancer unit hypothesis and
assess the relationships of enhancers to targeted promoters, and the role of specific motifs and sequence
features in enhancer function. In Aim 2, we examine quantitatively enhancers and key mutants identified in
Aim 1 by barcoding and integrating WT and mutant enhancers 5 kb upstream of their normally responsive
promoter in a chromosomal context at the AAVS1 safe harbor locus. These assays will rigorously test function
of enhancer motifs, core promoters, and the overall architecture of enhancers in a constant chromosomal
background. In addition, we will also test the regulatory code underlying enhancer specificity for promoters and
evaluate effects of mutant TF motifs on TF binding and on nascent transcription using targeted genomic
assays. Finally, in Aim 3, we explore the ability of enhancers to act over long distances, using the NMU
enhancer (eNMU), which resides 94 kb upstream of the NMU promoter and stimulates its transcription by
10,000-fold. We will utilize this robust model enhancer, which is not co...

## Key facts

- **NIH application ID:** 10878974
- **Project number:** 5R01HG012970-02
- **Recipient organization:** CORNELL UNIVERSITY
- **Principal Investigator:** JOHN T LIS
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $680,349
- **Award type:** 5
- **Project period:** 2023-07-01 → 2027-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10878974

## Citation

> US National Institutes of Health, RePORTER application 10878974, Functional Architecture and Interplay of Transcription Regulatory Elements of the Human Genome (5R01HG012970-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10878974. Licensed CC0.

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