# Genomics of Megakaryocyte and Platelet Biology

> **NIH NIH R01** · WASHINGTON UNIVERSITY · 2021 · $561,860

## Abstract

PROJECT SUMMARY
We recently found that mutations in ETV6 lead to thrombocytopenia with bleeding diathesis, red
cell macrocytosis, and predisposition to leukemia. We described families with missense mutations
in the central domain (p.Pro214Leu) and the ETS DNA binding domain (p.Arg418Gly) of ETV6
that result in aberrant cellular localization of ETV6, decreased transcriptional repression, and
impaired MK maturation. Deep sequencing of the platelet transcriptome revealed significant
differences in mRNA expression levels between patients with the ETV6 p.P214L mutation and
non-affected family members. Additionally, single cell RNA-sequence of peripheral mononuclear
blood cells from these patients demonstrated significant changes in the expression patterns of
mRNAs of Interferon (IFN) Response Genes, suggesting a critical role for ETV6 in maintaining
bone marrow homeostasis. This proposal will test the central hypothesis that normal regulation
and function of ETV6 is essential for transcriptional events that control MK differentiation and
formation of platelets that function properly under homeostatic and inflammatory conditions. We
generated a transgenic mouse in which Etv6 exhibits the p.P214L mutation at the mouse
orthologue conserved position (Etv6P214L). Mice with this mutation (Etv6P214L) have reduced
platelet counts and exhibit a platelet defect. In this proposal, we will test three aims that will
determine the mechanisms by which ETV6 regulates critical functions of MKs and platelets. In
Specific Aim 1 we will define roles for ETV6 in regulating MK differentiation, platelet formation
and platelet function. For this purpose, we will use Etv6P214L, Etv6-/- Gata1-Cre and Etv6-/- Pf4-Cre
mice to determine the effects of Etv6 gene disruption in MK progenitors and MKs. These results
will be compared to MK differentiation and proplatelet formation in CD34+- derived MK that are
cultured from patients with ETV6 mutations. Additionally, we will study in vitro and in vivo platelet
responses from these mice. In Specific Aim 2 we will delineate the contributions of ETV6 in
modulating transcriptional events in MK. This aim will test the hypothesis that ETV6 directly
regulates transcriptional events in MKs. We will identify Etv6-dependent gene candidates by
performing RNA-seq in MKs and platelets isolated from Etv6P214L, Etv6-/-Gata1-Cre, and Etv6-/-
Pf4-Cre mice. These results will be compared to Chip-seq/DNase I seq data and results obtained
from mice and human MKs that carry the ETV6 p.P214L and R418G mutations. We will determine
the mechanisms by which ETV6 regulates the transcription of candidate mRNAs by identifying
ETV6 effectors and test them functionally. Finally, in Specific Aim 3 we will determine the
consequences of ETV6 disruption on IFN response genes in bone marrow homeostasis. This aim
will test the hypothesis that ETV6 regulates IFN response genes by interacting with HDAC3, and
disruption of ETV6 function will generate a proinflammatory milie...

## Key facts

- **NIH application ID:** 10089473
- **Project number:** 5R01HL139825-02
- **Recipient organization:** WASHINGTON UNIVERSITY
- **Principal Investigator:** Jorge A Di Paola
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $561,860
- **Award type:** 5
- **Project period:** 2020-02-01 → 2024-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10089473

## Citation

> US National Institutes of Health, RePORTER application 10089473, Genomics of Megakaryocyte and Platelet Biology (5R01HL139825-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10089473. Licensed CC0.

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