# Decoding the gene regulatory landscape of pluripotency and differentiation

> **NIH NIH P01** · HARVARD UNIVERSITY · 2020 · $610,158

## Abstract

Abstract: 
Over the last five years, the characterization of the pluripotent state and its differentiated progeny has made 
remarkable progress. Previous studies have identified most of the building blocks participating in the 
establishment, maintenance and differentiation of the pluripotent state. However, most studies so far relied on 
correlation based evidence and inference, and did not yet obtain a functionally validated picture of its 
regulatory foundation. Here, we propose to overcome parts of this problem by addressing the following 
questions: 
Aim 1: What are the functionally relevant cis-regulatory modules (CRMs) and gene regulatory networks 
(GRNs) for pluripotency and differentiation initiation? Previous work has mapped the epigenetic state, 
transcription factor binding and RNA expression in pluripotent, reprogramming and differentiating cells, yielding 
a list of putative gene regulatory elements (GREs). Here, we will define the subset of functionally relevant 
GREs and CRMs for these cell types. To that end, we will employ massively parallel reporter assays (MPRA) 
to assess the functionality of thousands of GREs in parallel, identify their upstream transcriptional regulators 
through targeted motif mutagenesis and define their phenotypic relevance through epigenome editing 
mediated loss of function experiments for hundreds of GREs. 
Aim 2: What functional role does DNA methylation play in the regulation of CRMs in pluripotency and 
differentiation? While it is well established that changes in the DNA methylation patterns can demarcate GREs, 
it is less clear whether these changes are functionally relevant for the proper operation of the corresponding 
CRMs. To investigate the contextual relevance of DNA methylation changes, we will employ a modified version 
of our MPRA that allows for the identification of those GREs that operate in a methylation sensitive fashion. 
These experiments will yield insights into context specific role of DNA methylation in the control of cell type 
specific GRNs. 
Aim 3: What is the cis-regulatory code controlling long non-coding RNA (lncRNA) activity and localization in 
pluripotency and differentiation? Recent findings from the Project II of this proposal identified a set of lncRNAs 
critical for pluripotency and differentiation. However, their regulation on the DNA and RNA level remains 
elusive. In order to address this question, we will employ two distinct novel MPRA designs, one for DNA 
elements and one adapted to RNA elements. These experiments will define the cis-regulatory landscape of 
lncRNA regulation on the DNA and RNA level in pluripotency and differentiation.

## Key facts

- **NIH application ID:** 9959201
- **Project number:** 5P01GM099117-09
- **Recipient organization:** HARVARD UNIVERSITY
- **Principal Investigator:** Andreas Gnirke
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $610,158
- **Award type:** 5
- **Project period:** — → —

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9959201

## Citation

> US National Institutes of Health, RePORTER application 9959201, Decoding the gene regulatory landscape of pluripotency and differentiation (5P01GM099117-09). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9959201. Licensed CC0.

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