# Transcriptional response to signaling during hematopoiesis

> **NIH NIH R01** · BOSTON CHILDREN'S HOSPITAL · 2021 · $532,262

## Abstract

ABSTRACT
Hematopoietic differentiation involves progression from the progenitor to precursor stages, and final
maturation. Master Transcription factors (MTFs) such as GATA1 and GATA2 activate a critical cell-specific
program, but additional transcription factors that drive stage-specific expression remain to be defined.
Extracellular signals are transmitted to the nucleus, which activate signaling transcription factors (STFs). We
studied human CD34 cells differentiated to the erythroid lineage, and examined the activation and binding of
specific STFs to DNA representing several signaling pathways. We identified regions of the genome
corresponding to stage-specific genes that are co-occupied by MTFs and STFs. We called these co-occupied
regions “transcriptional signaling centers” (TSCs) because they render the adjacent genes inducible by growth
factors or small molecules. The BMP-signaling transcription factor SMAD1 is a marker of active TSCs and
binds adjacent to GATA-factors to mark active genes at each stage of differentiation. SMAD1 is predictive of
where other STFs bind, such as the cAMP-directed CREB, WNT-directed TCF7L2, and TGFβ-directed
SMAD2. Each ligand can activate (or repress) TSCs, leading to altered enhancer activity and gene expression.
Co-binding of SMAD1 and GATA factors allows BMP induction of target genes, and mutation of a SMAD1-site
in one TSC demonstrated a requirement of SMAD1-binding for appropriate gene expression. An examination
of single nucleotide polymorphisms (SNPs) associated with erythroid traits demonstrates enrichment of such
variations at TSCs, where many mutations occur at SMAD or other STF binding sites within the local region.
The majority of human erythroid GWAS genes have mutations in STF binding sites in TSCs, but only a minority
of SNPs affect the binding of MTFs. We showed that a polymorphism in a SMAD binding site within a TSC
reduces SMAD1 binding based on gel mobility shift analysis and causes a specific reduction of expression of
the associated gene in human blood cells. Other signals such as PGE2 also lead to activation of TSCs. We
have shown that PGE2 induces stem cell birth during embryogenesis, and enhances hematopoietic stem cell
(HSC) transplantability in fish, mice and humans. PGE2 enhanced HSCs are currently in a fourth clinical trial
for patients with leukemia. Since the PGE2-stimulated STF CREB binds adjacent to SMAD1 in TSCs, we plan
to examine if targets of both pathways are similar, or if specific gene programs are activated according to
ligands. We will evaluate how PGE2 and BMP alter chromatin to lead to specific gene expression changes. Our
data using micrococcal nuclease sensitivity studies suggest that within a few hours, there is a reorganization of
chromatin resulting in greater accessibility of regions bound by the STFs. We plan to utilize ChIP-seq, ATAC-
seq and protein biochemistry to examine how these chromatin alterations lead to gene expression changes.
Understanding the spe...

## Key facts

- **NIH application ID:** 10072076
- **Project number:** 5R01HL144780-03
- **Recipient organization:** BOSTON CHILDREN'S HOSPITAL
- **Principal Investigator:** LEONARD Ira ZON
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $532,262
- **Award type:** 5
- **Project period:** 2019-01-15 → 2022-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10072076

## Citation

> US National Institutes of Health, RePORTER application 10072076, Transcriptional response to signaling during hematopoiesis (5R01HL144780-03). Retrieved via AI Analytics 2026-06-11 from https://api.ai-analytics.org/grant/nih/10072076. Licensed CC0.

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