# Mechanisms of Transcriptional Control Revealed by Nascent Transcript Sequencing

> **NIH NIH R01** · HARVARD MEDICAL SCHOOL · 2024 · $580,405

## Abstract

Transcription initiation is highly regulated by genomic elements in promoters and enhancers that bind
transcription factors and recruit RNA polymerase II (Pol II). After Pol II escapes from the promoter, many
regulatory steps occur during transcription elongation that control Pol II progression, RNA processing, and
finally release of the RNA from chromatin for nuclear export. These steps are tightly controlled by genomic and
epigenomic features, such as histone modifications, nucleosome positions, and cis-elements that bind
transcription factors and RNA-binding proteins (RBPs). Current functional genomic technologies tend to
provide a static, averaged view of genomic function that obscure the transient roles that many genomic
elements play in shaping the transcriptome. To identify and characterize all functional genomic elements, we
need to develop approaches that quantitatively monitor the many layers of gene expression regulation,
including transient interactions and short-lived events. In the proposed research, we will create a suite of
genome-wide tools to map Pol II across single chromatin fibers, track the association of RBPs, and follow
RNAs from transcription initiation to nuclear export. These techniques will highlight unappreciated connections
between genomic elements, molecular events on chromatin, and post-initiation regulatory events. Our rationale
is that by creating tools to quantify short-lived steps in mRNA production, unappreciated functional roles for
genomic elements can be revealed. To demonstrate how these technologies reveal unappreciated gene
regulatory mechanisms, we will apply them to the heat shock response (HSR), a stress response that has
served as a model to study the many regulatory layers in gene expression. In Aim 1 we will develop a strategy
that reveals Pol II locations across single long chromatin fibers. Fiber-seq is a method that identifies regions of
accessible DNA across long (up to ~20 kb) chromatin fibers one molecule at a time. We will extend this
approach to identify Pol II footprints, analyze the distributions of Pol II complexes across gene bodies, and
characterize the interactions between Pol II complexes and nucleosomes. In Aim 2 we will measure the timing
of binding of proteins across long pre-mRNA molecules. RBP binding will be marked by RNA editing and long-
read direct RNA sequencing of nascent RNA (nano-COP) will enable us to resolve transient binding events
that impact pre-mRNA processing and nuclear export. These events are transient, as many RBPs bind to
introns and are released from RNA in the cytoplasm immediately after nuclear export. In Aim 3, we will create
an approach to measure kinetics of RNA production, chromatin release, and nuclear export that couples 4sU
metabolic labeling of newly synthesized RNAs with biochemical purification of RNAs from chromatin,
nucleoplasm, and cytoplasm. The data from this technique will allow us to estimate the rates of release of RNA
from chromatin, nuc...

## Key facts

- **NIH application ID:** 10770481
- **Project number:** 5R01HG007173-11
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** Lee Stirling Churchman
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $580,405
- **Award type:** 5
- **Project period:** 2013-04-01 → 2027-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10770481

## Citation

> US National Institutes of Health, RePORTER application 10770481, Mechanisms of Transcriptional Control Revealed by Nascent Transcript Sequencing (5R01HG007173-11). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10770481. Licensed CC0.

---

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