ETV6 germline mutations cause HDAC3/NCOR2 mislocalization and upregulation of interferon response genes
Marlie H. Fisher, Gregory D. Kirkpatrick, Brett Stevens, Courtney Jones, Michael Callaghan, Madhvi Rajpurkar, Joy Fulbright, Megan A. Cooper, Jesse Rowley, Christopher C. Porter, Arthur Gutierrez-Hartmann, Kenneth Jones, Craig Jordan, Eric M. Pietras, Jorge Di Paola
Marlie H. Fisher, Gregory D. Kirkpatrick, Brett Stevens, Courtney Jones, Michael Callaghan, Madhvi Rajpurkar, Joy Fulbright, Megan A. Cooper, Jesse Rowley, Christopher C. Porter, Arthur Gutierrez-Hartmann, Kenneth Jones, Craig Jordan, Eric M. Pietras, Jorge Di Paola
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Research Article Hematology

ETV6 germline mutations cause HDAC3/NCOR2 mislocalization and upregulation of interferon response genes

Abstract

ETV6 is an ETS family transcription factor that plays a key role in hematopoiesis and megakaryocyte development. Our group and others have identified germline mutations in ETV6 resulting in autosomal dominant thrombocytopenia and predisposition to malignancy; however, molecular mechanisms defining the role of ETV6 in megakaryocyte development have not been well established. Using a combination of molecular, biochemical, and sequencing approaches in patient-derived PBMCs, we demonstrate abnormal cytoplasmic localization of ETV6 and the HDAC3/NCOR2 repressor complex that led to overexpression of HDAC3-regulated interferon response genes. This transcriptional dysregulation was also reflected in patient-derived platelet transcripts and drove aberrant proplatelet formation in megakaryocytes. Our results suggest that aberrant transcription may predispose patients with ETV6 mutations to bone marrow inflammation, dysplasia, and megakaryocyte dysfunction.

Authors

Marlie H. Fisher, Gregory D. Kirkpatrick, Brett Stevens, Courtney Jones, Michael Callaghan, Madhvi Rajpurkar, Joy Fulbright, Megan A. Cooper, Jesse Rowley, Christopher C. Porter, Arthur Gutierrez-Hartmann, Kenneth Jones, Craig Jordan, Eric M. Pietras, Jorge Di Paola

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Figure 2

HDAC3/NCOR2 complex binds to ETV6 and is mislocalized in ETV6 P214L and R369Q PBMC, resulting in increased acetylation.

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HDAC3/NCOR2 complex binds to ETV6 and is mislocalized in ETV6 P214L and ...
(A) Comparison via immunofluorescence microscopy of HDAC3 localization in PBMCs from ETV6 P214L and R369Q patients and healthy controls. Representative PBMCs stained for DNA (blue) and HDAC3 (red). In cells from healthy controls, HDAC3 is concentrated in the nucleus. In contrast, cells from patients carrying ETV6 P214L or R369Q demonstrated HDAC3 concentrated in the cytoplasm, with scarce staining in the nucleus. Scale bar: 20 μm. Original magnification, ×8 (inset). (B) Nuclear-to-cytoplasmic intensity ratio of HDAC3 is significantly reduced in ETV6 P214L PBMCs (unpaired t test, **P = 0.0071). Fluorescence intensity was calculated in the nucleus and cytoplasm. n = 5 random fields of view were used to calculate N/C intensity ratio, representing a minimum of 100 cells analyzed in each condition. (C) Comparison via immunofluorescence microscopy of NCOR2 localization in PBMCs from ETV6 P214L patients and healthy controls. Representative PBMCs stained for DNA (blue) and cofactors listed above (red). In cells from healthy controls, NCOR2 is concentrated in the nucleus. In contrast, cells from patients carrying ETV6 P214L and R369Q demonstrated NCOR2 concentrated in the cytoplasm, with scarce staining in the nucleus. Scale bar: 20 μm. Original magnification, ×8 (inset). (D) Nuclear-to-cytoplasmic intensity ratio of NCOR2 is significantly reduced in ETV6 P214L PBMCs (unpaired t test, *P = 0.0169). Fluorescence intensity was calculated in the nucleus and cytoplasm. n = 5 random fields of view were used to calculate N/C intensity ratio, representing a minimum of 100 cells analyzed in each condition. (E) Immunoprecipitation of ETV6 in healthy control PBMCs demonstrates ETV6 interacting with HDAC3 and NCOR2. α-Tubulin was included to demonstrate equal loading of protein in each sample. Representative image of 2 independent experiments. (F) Transient knockdown of ETV6 by siRNA in healthy control PBMCs results in increased acetylated histone H3 compared with control conditions. Representative image of 2 independent experiments. (G) CRISPR-mediated knockout of ETV6 in cord blood–derived CD34+ HSCs drives increased acetylation of histone H3. Representative image of 2 independent experiments.