ETV6 knockout leads to impaired megakaryoid gene regulation, which is phenocopied by the ETS-domain mutant ETV6R369W via dominant negative actions
Megakaryopoiesis is a process where haematopoietic stem cells differentiate to megakaryocytes and platelets. This process is controlled by temporal expression of different transcription factors. TEL/ETV6 is an ETS family transcription factor that recently has been discovered to be associated with autosomal dominant thrombocytopenia. ETV6 mainly functions as a transcriptional repressor through dimerisation via pointed (PNT) domain and interactions with other cofactors via the central domain. DNA binding is via the conserved ETS domain, where most clinically relevant mutations are located. Here we aimed to unravel the role of ETV6 and patient-specific mutants thereof in megakaryopoiesis.
To understand the role of ETV6 in megakaryopoiesis, RNA sequencing and mass spectrometry were conducted on ETV6 knock out (KO) clones generated using megakaryoblast resembling Meg01 cells. ETV6WT or ETV6R369W was introduced to Meg01 cells with wild type or knock out background, followed by RNA sequencing and mass spectrometry. ETV6WT or ETV6R369W was introduced to ETV6 KO clones with v5-ETV6WT or v5-ETV6R369W, followed by immunoprecipitation using anti-v5 antibody to study ETV6 WT and mutant interaction. ETV6WT or ETV6R369W was also introduced to ETV6 KO clones alone, and immunoprecipitation was performed using anti-ETV6 antibody to study cofactor interactions of the mutant.
Consistent with the repressive function of ETV6, RNA sequencing analysis of ETV6 knockout cells revealed 507 upregulated and 292 downregulated genes compared to wildtype cells. Erythroid genes GYPA, FUT1 and ALAS2, were downregulated, putatively caused by increased expression of FLI1, a critical megakaryopoiesis driver. Signalling pathways genes established in stem cell identity, such as KIT, CD44, IL1R, IL6R, were upregulated and validated by flow cytometry. A conflict in cellular identity may contribute to abnormal megakaryocyte differentiation and maturation. Increased expression of several genes was confirmed by proteomics in platelets of patients bearing ETV6R369W mutation. Lentiviral expression of ETV6R369W in wild-type Meg-01 clones resulted in decreased GYPA expression, with levels comparable to ETV6 knockout clones. Co-immunoprecipitation experiments indicated an interaction between ETV6WT and ETV6R369W and similar cofactors binding, suggestive of dimerization and a possible basis of the dominant negative action.
In conclusion, here we find that ETV6 knockout leads to a significant deregulation of genes during megakaryopoiesis, while the data suggests a dominant negative mode of action for ETV6R369W. The mechanistic and molecular insights allow better understanding of the ETV6-mutant-induced thrombocytopenia.