# Molecular Analysis of Transcriptional Enhancers in Hematopoiesis

> **NIH NIH R01** · UT SOUTHWESTERN MEDICAL CENTER · 2020 · $364,500

## Abstract

PROJECT SUMMARY
Elucidating mechanisms that regulate gene expression in red blood cell production is fundamental to
understanding cellular differentiation, and to developing new therapies for red cell disorders. Transcriptional
enhancers determine cell identity by directing spatiotemporal gene expression. Recently, we and others have
identified erythroid-specific enhancer elements through genome-scale profiling of chromatin features. Further
analysis has uncovered GATA1-interacting coactivators and their combinations as candidate drivers of
enhancer function. These studies established a comprehensive catalog of erythroid enhancer elements, yet the
molecular composition and in vivo function of the vast majority of these enhancers remain unknown. Given that
enhancers are frequently targeted by disease-associated genetic variations, it is imperative to fill this gap in
knowledge. The objective of this project is to determine the protein complexes and long-range DNA
interactions responsible for enhancer assembly and function in situ during erythropoiesis. The central
hypothesis is that erythroid enhancers are assembled by specific combinations of lineage-regulating
transcription factors such as GATA1 to recruit transcriptional coactivators and to initiate long-range chromatin
interactions for gene transcription. This hypothesis has been formulated on the basis of our preliminary studies
of an erythroid-specific super-enhancer comprised of three individual enhancers that regulates the SLC25A37
gene, a mitochondrial iron transporter essential for iron metabolism and heme synthesis, and an innovative
approach to identify macromolecules associated with a single genomic locus using the endonuclease-deficient
Cas9 (dCas9) and single guide RNA (sgRNA) components of the CRISPR system. Guided by these
preliminary data, this hypothesis will be tested by pursuing three specific aims: 1) Identify and characterize
SLC25A37 enhancer-associated proteins in situ by dCas9 affinity purification, followed by quantitative
proteomic analysis and validation studies. 2) Determine SLC25A37 enhancer long-range DNA interactions
using dCas9-mediated chromatin interaction analysis by paired-end sequencing (dCas9-ChIA-PET). By
comparing the frequency and location of chromatin interactions between individual SLC25A37 enhancers and
their temporal changes during erythropoiesis, these analyses will establish functional links between chromatin
looping and enhancer function. 3) Define the functional requirement of 41 erythroid disease-associated,
evolutionarily conserved super-enhancers using an engineered dCas9-LSD1 repressor complex in a dCas9
knockin mouse model. Together these studies will not only elucidate the mechanisms for the genetic control of
a principal regulator of erythroid iron metabolism, but also provide new tools for in situ analysis of enhancer-
regulating components. Such results are expected to advance our understanding of the composition and
function of enhancers...

## Key facts

- **NIH application ID:** 9995472
- **Project number:** 5R01DK111430-05
- **Recipient organization:** UT SOUTHWESTERN MEDICAL CENTER
- **Principal Investigator:** Jian Xu
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $364,500
- **Award type:** 5
- **Project period:** 2016-09-19 → 2021-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9995472

## Citation

> US National Institutes of Health, RePORTER application 9995472, Molecular Analysis of Transcriptional Enhancers in Hematopoiesis (5R01DK111430-05). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9995472. Licensed CC0.

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