Molecular mechanisms of bleeding disorderassociated GFI1B(Q287*) mutation and its affected pathways in megakaryocytes and platelets

Dominant-negative mutations in the transcription factor Growth Factor Independence-1B (GFI1B), such as GFI1B(Q287*), cause a bleeding disorder characterized by a plethora of megakaryocyte and platelet abnormalities. The deregulated molecular mechanisms and pathways are unknown. Here we show that both normal and Q287* mutant GFI1B interacted most strongly with the lysine specific demethylase-1 – REST corepressor - histone deacetylase (LSD1-RCOR-HDAC) complex in megakaryoblasts. Sequestration of this complex by GFI1B(Q287*) and chemical separation of GFI1B from LSD1 induced abnormalities in normal megakaryocytes comparable to those seen in patients. Megakaryocytes derived from GFI1B(Q287*)-induced pluripotent stem cells also phenocopied abnormalities seen in patients. Proteome studies on normal and mutant-induced pluripotent stem cell-derived megakaryocytes identified a multitude of deregulated pathways downstream of GFI1B(Q287*) including cell division and interferon signaling. Proteome studies on platelets from GFI1B(Q287*) patients showed reduced expression of proteins implicated in platelet function, and elevated expression of proteins normally downregulated during megakaryocyte differentiation. Thus, GFI1B and LSD1 regulate a broad developmental program during megakaryopoiesis, and GFI1B(Q287*) deregulates this program through LSD1-RCOR-HDAC sequestering.