# Towards fully reconstituting mammalian transcription in a test tube

> **NIH NIH DP2** · MASSACHUSETTS INSTITUTE OF TECHNOLOGY · 2021 · $1,303,608

## Abstract

Project Summary/Abstract
The human genome measures nearly 2 meters in length and must be compacted to fit in the nucleus, which
measures only a few microns in diameter. In addition to compaction, the DNA must be organized to maintain
genome integrity but also remain accessible to the molecular machines that read the genome. How the
genome is organized directly influences which genes are expressed in a particular cell. Decoupling of genome
organization and gene expression has profound consequences for cells, leading to cancer, intellectual
disability, and developmental delay. It is mechanistically unclear how genome organization and the first step of
gene expression, transcription, are physically coupled. Determining how both processes are linked is critical for
understanding how cell type function and specificity are achieved. The first level of eukaryotic genome
compaction and organization is mediated by nucleosomes that are formed by wrapping DNA around histone
proteins. Here I will investigate how DNA sequence and nucleosomes impact gene expression in promoter
proximal regions of human genes. I will mechanistically decipher how the processes of transcription and
genome compaction are intertwined by reconstituting transcription reactions on chromatin in vitro and
observing them using (1) a novel high throughput biochemical assay and (2) time-resolved cryo-electron
microscopy (EM).
 In the first part of this proposal, we will develop a high throughput, single molecule transcription assay
using reconstituted transcription complexes on thousands of DNA sequences. We will assess how DNA
sequence, chromatin state, and transcription factors directly regulate transcriptional activity during early
transcription elongation. This assay will overcome major hurdles in the field by revealing directly how
nucleosomes and protein factors affect transcription behavior on an unprecedented number of DNA sequence
contexts.
 In the second part of this proposal, we will define how chromatin and DNA sequence influence
transcription dynamics by capturing structural snapshots of transcription complexes as they transcribe through
nucleosomes. We will use time-resolved cryo-EM to directly assess which states transcription complexes adopt
during early transcription and identify stable online and labile offline states. Together, this ambitious proposal
will address important questions regarding how nucleosomes and DNA sequence are used to directly influence
transcriptional output. The methods and data acquired in this proposal will help fulfill the long-term vision of my
lab to fully reconstitute mammalian transcription to determine how cell fate is regulated by the coupling of
genome organization and gene expression and how disease mutations can perturb this coupling.

## Key facts

- **NIH application ID:** 10242352
- **Project number:** 1DP2GM146254-01
- **Recipient organization:** MASSACHUSETTS INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Seychelle Monqiue Vos
- **Activity code:** DP2 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $1,303,608
- **Award type:** 1
- **Project period:** 2021-09-21 → 2024-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10242352

## Citation

> US National Institutes of Health, RePORTER application 10242352, Towards fully reconstituting mammalian transcription in a test tube (1DP2GM146254-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10242352. Licensed CC0.

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