# Regulatory Roles and Dynamics of Nuclear long-noncoding RNAs in Pluripotency

> **NIH NIH P01** · HARVARD UNIVERSITY · 2021 · $578,655

## Abstract

One of the biggest surprises of the human genome project was the vast amount of transcription observed in 
the noncoding genome, comprised of thousands of long noncoding RNAs (lncRNA). In previous work, we 
identified a few lncRNAs that contribute important functional roles in human reprograming and maintenance of 
pluripotency. Our previous proposal, in collaboration with the Drs. Meissner's and Gnirke's proposals, has now 
identified more than a hundred lncRNAs that are specific to the pluripotent state, dynamically regulated during 
reprograming and enriched to reside near key developmental regulators. We have also identified an emerging 
mechanistic theme of lncRNAs as regulatory factors in shaping nuclear architecture and, in turn, driving 
pluripotent gene expression programs. Based on these studies we now propose to take the next leap forward 
towards addressing the following pressing questions: 
Aim 1: Do pluripotent specific expressed lncRNA loci that are in proximity to pluripotency and developmental 
regulators functionally contribute to the maintenance of pluripotency? It is tempting to think that lncRNAs that 
reside near key pluripotency and developmental regulators may function in cis to either activate pluripotency 
factors or repress differentiation genes. Here we will systematically apply a transcriptional inhibition (CRISPR-i) 
screen on a hundred of these lncRNA loci for those that are required to maintain pluripotency. We will further 
perform numerous individual validation tests and RNase-H mediated depletion of lncRNAs in parallel. Finally, 
we will determine influences on pluripotent and differentiation gene-expression programs upon lncRNA loss-of 
function (LOF) using massively-parallel RNA-sequencing technologies. 
Aim 2: What are the genome wide RNA-DNA and RNA-Protein localization properties of nuclear lncRNAs that 
facilitate proper pluripotency gene-expression programs? We have revised the proposal for a deeper focus on 
FIRRE and CISTR-ACT, two lncRNAs that, as determined in the previous proposal, share key properties highly 
relevant to the proposed study: required for stemness, shared mechanism of facilitating nuclear architecture 
and strongly linked to human disease. By performing loss of function studies on identified protein partners we 
can begin to determine the underlying influences of RNA/DNA sequences, chromatin environments, three- 
dimensional proximity and protein interactions on lncRNA nuclear localization. 
Aim 3: What are the dynamics of FIRRE and CISTR-ACT mediated nuclear organization. We now have 
adapted CRISPR-Display to Live Cell Imaging (CLING). With this approach we can monitor the 3D interactions 
of up to 5 chromosomes through time in a living cell. We will investigate the WT and LOF and GOF states of 
FIRRE and CISTR-ACT chromosomal dynamics in pluripotency and during reprograming. Collectively, these 
studies will identify the molecular interactions of lncRNAs with DNA and Protein and ho...

## Key facts

- **NIH application ID:** 10144472
- **Project number:** 5P01GM099117-10
- **Recipient organization:** HARVARD UNIVERSITY
- **Principal Investigator:** John Louis Rinn
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $578,655
- **Award type:** 5
- **Project period:** 2011-08-01 → 2023-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10144472

## Citation

> US National Institutes of Health, RePORTER application 10144472, Regulatory Roles and Dynamics of Nuclear long-noncoding RNAs in Pluripotency (5P01GM099117-10). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10144472. Licensed CC0.

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