# H3.3-mediated epigenetic regulation of developmental bivalent genes for reprogramming and differentiation

> **NIH NIH R01** · WEILL MEDICAL COLL OF CORNELL UNIV · 2020 · $355,950

## Abstract

PROJECT SUMMARY: Reprogramming somatic cells to become pluripotent stem cells holds
great promise for stem-cell-based therapeutics, but one of the major barriers lies in the ability to
identify which of the stem cells are truly pluripotent. We have shown that induced pluripotent
stem cells (iPSCs) from mice can have markedly different potential to generate “all-iPS”
animals, even when the cells have similar transcription profiles. This difference suggests an
essential role for epigenetic mechanisms in regulating stem cell pluripotency. When
developmental bivalent (DB) genes are activated in lineage commitment, they are silenced but
poised for later activation in pluripotent stem cells. We currently have no way to probe for this
necessary poised state, as transcriptional profiles of iPSCs do not reveal potential for
transcriptional activation. We previously reported that H3.3 is required to establish pluripotency
during reprogramming and is required to maintain the bivalency of DB genes in ESCs. In fact,
our preliminary data showed that H3.3 is enriched at the promoter of many DB genes, and its
lack of enrichment correlates tightly with compromised developmental potential in iPSCs. This
finding indicates that H3.3 plays a critical role in regulating DB gene expression, and thus
pluripotency. Based on our observations, we hypothesize that H3.3 is required to establish
bivalency during reprogramming and that this epigenetic signature at the promoter poises DB
genes for later activation upon differentiation. Our long-term goal is to elucidate the key
mechanisms of establishing and maintaining pluripotency in stem cells. The objective of this
proposal is to define the mechanisms by which the histone variant H3.3 regulates the DB genes
and the developmental properties of stem cells. We plan to test the hypothesis using unique
animal models that will permit us to obtain enough genetically uniform cells at any intermediate
stage. This will allow us to study both H3.3 and histone modifications using ChIP sequencing
during reprogramming. We propose the following two aims in this application: Aim 1: Identify
how H3.3 regulates the establishment of epigenetic signatures in DB genes during
reprogramming toward pluripotency. Aim 2: Determine the function of H3.3 enrichment mark at
the promoter in DB genes during iPSC differentiation. Successful completion of these aims will
allow us to identify how the enrichment of H3.3 at the promoter for DB genes impacts the
potential for differentiation of stem cells into specific cell lineages. This work will provide both
key information on the functional relevance of epigenetic regulation of pluripotency, and also a
possible clinical approach to evaluate the pluripotency of stem cells.

## Key facts

- **NIH application ID:** 9955280
- **Project number:** 5R01GM129380-03
- **Recipient organization:** WEILL MEDICAL COLL OF CORNELL UNIV
- **Principal Investigator:** Duancheng Wen
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $355,950
- **Award type:** 5
- **Project period:** 2018-08-01 → 2023-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9955280

## Citation

> US National Institutes of Health, RePORTER application 9955280, H3.3-mediated epigenetic regulation of developmental bivalent genes for reprogramming and differentiation (5R01GM129380-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9955280. Licensed CC0.

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