# Project 4 Functional Dissection of Erythroid Super-Enhancer, Daniel E. Bauer

> **NIH NIH P01** · BOSTON CHILDREN'S HOSPITAL · 2021 · $317,577

## Abstract

ABSTRACT
 Enhancers are non-coding DNA elements near genes that positively regulate gene expression.
Because enhancers often act in a cell type specific manner, they confer biological specificity on gene
regulation. Common genetic variation within enhancers appears to be a prevalent means of trait association.
Super-enhancers are a recently described set of enhancers highly enriched for regulatory elements of cell
lineage-defining genes and genetic variants related to lineage-specific disease susceptibility. However the
structural determinants of super-enhancer function remain poorly understood. Enhancers are typically defined
by their correlated biochemical features or ectopic potential in reporter assays. Nonetheless the gold-standard
test of enhancer activity is loss-of-function mutation to discern the requirement for regulatory sequences in
their natural chromosomal environment. Our group has developed a Cas9-mediated in situ saturating
mutagenesis technique that allows for high-resolution, high-throughput functional analysis of enhancers in the
native genomic setting. The assay involves design and synthesis of guide RNAs saturating a region of interest
followed by pooled lentiviral CRISPR screening. This method when coupled with genomic target deep
sequencing allows for an evaluation nearing nucleotide resolution of the functional sequences critical for
enhancer function. In this proposal, we apply this technique to systematically perturb twenty erythroid-specific
super-enhancers of key erythroid genes. We extend the Cas9 mutagenesis strategy by making use of a variant
Cas9 to allow for increased genome editing resolution. In addition, we take a variant-informed guide design
approach to improve mutagenesis even in the face of natural genomic variation. We utilize bioinformatic
methods to identify essential sequences, predict interacting transcription factors, and refine models to estimate
enhancer activity. We use biochemical techniques to determine the occupancy of transcription factors and
chromatin regulators at required sequences. We focus on defining and testing super-enhancer looping
interactions in which the key transcription factors GATA1 and TAL1 participate. Finally we employ prospective
reverse genetics to validate both cis-acting sequences and trans-acting factors necessary for super-enhancer
function. These studies will help determine emergent cooperative properties of clustered components within
super-enhancers. We expect these experiments to inform a further understanding of crucial aspects of
erythropoiesis as well as fundamental mechanisms of gene regulation.

## Key facts

- **NIH application ID:** 10146453
- **Project number:** 5P01HL032262-39
- **Recipient organization:** BOSTON CHILDREN'S HOSPITAL
- **Principal Investigator:** Daniel Evan Bauer
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $317,577
- **Award type:** 5
- **Project period:** 1997-07-01 → 2022-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10146453

## Citation

> US National Institutes of Health, RePORTER application 10146453, Project 4 Functional Dissection of Erythroid Super-Enhancer, Daniel E. Bauer (5P01HL032262-39). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10146453. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*