# Characterizing global regulatory networks in human embryonic stem cells

> **NIH NIH F30** · YALE UNIVERSITY · 2020 · $47,688

## Abstract

Project Summary: Characterizing global regulatory networks in human embryonic stem cells
Human embryonic stem cells (hESCs) provide a model for early preimplantation development and have the
potential to be harnessed for applications in regenerative medicine. However, a comprehensive, integrated
view of the regulatory network underlying hESC identity is fundamentally lacking. The overarching goal of this
proposal is to elucidate novel features of the global regulatory architecture in hESCs. In order to discover
critical components of this network, a genome-wide shRNA screen was performed, identifying a number of
previously unrecognized transcription factors and epigenetic modifiers essential to hESC biology, such as
EP400, an epigenetic modifier that notably acts as a transcriptional activator in other cell types. Screen hits
were characterized and narrowed to a final shortlist of 15 novel genes with a clear role in hESC maintenance.
These factors will be sorted into functional categories and organized into wider, integrated clusters of co-
regulation within the hESC network.
The first aim will seek to characterize this newly defined set of novel, screen-identified factors alongside
known, established hESC factors. Loss-of-function experiments will be used to sort each gene into functional
categories defined by knockdown effects on pluripotency, self-renewal, or viability. Further characterization of
gene function in hESCs will be conducted using functional assays and analysis of molecular markers.
The second aim will employ global deep-sequencing-based approaches to identify relationships between
critical hESC factors and reconstruct co-regulatory modules important to the maintenance of hESCs.
Transcription factors will be clustered by their impact on global expression profile upon knockdown, with
patterns of expression changes shared by co-regulators. Binding site analysis will allow for specific mapping of
the regulatory circuitry that establishes and maintains the hESC transcriptional landscape. Finally, the same
profiling methods will be used to integrate EP400 and its associated histone variant H2A.Z into this network to
determine whether the structure of this epigenetic pathway aligns with downstream transcriptional modules as
hypothesized, or displays its own unique, independent organization.
The proposed studies will thus provide a systems view of the mechanisms by which transcriptional and
epigenetic regulators converge to drive the unique programs of pluripotency and self-renewal in the early
human embryo. A better understanding of the complex, network-based regulation governing hESC behavior
will help address the need for more accessible models of human development and guide the advancement of
regenerative therapies such as somatic cell reprogramming.

## Key facts

- **NIH application ID:** 9844862
- **Project number:** 5F30HD093350-03
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Alice Lu
- **Activity code:** F30 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $47,688
- **Award type:** 5
- **Project period:** 2018-01-16 → 2021-01-15

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9844862

## Citation

> US National Institutes of Health, RePORTER application 9844862, Characterizing global regulatory networks in human embryonic stem cells (5F30HD093350-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9844862. Licensed CC0.

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