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Molecular mechanisms of bleeding disorderassociated GFI1BQ287* mutation and its affected pathways in megakaryocytes and platelets.

ABSTRACT: Dominant-negative mutations in the transcription factor Growth Factor Independence-1B (GFI1B), such as GFI1BQ287*, 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 GFI1BQ287* and chemical separation of GFI1B from LSD1 induced abnormalities in normal megakaryocytes comparable to those seen in patients. Megakaryocytes derived from GFI1BQ287*-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 GFI1BQ287* including cell division and interferon signaling. Proteome studies on platelets from GFI1BQ287* 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 GFI1BQ287* deregulates this program through LSD1-RCOR-HDAC sequestering.

SUBMITTER: van Oorschot R

PROVIDER: S-EPMC6601108 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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Molecular mechanisms of bleeding disorderassociated GFI1B<sup>Q287*</sup> mutation and its affected pathways in megakaryocytes and platelets.

van Oorschot Rinske R Hansen Marten M Koornneef Johanna M JM Marneth Anna E AE Bergevoet Saskia M SM van Bergen Maaike G J M MGJM van Alphen Floris P J FPJ van der Zwaan Carmen C Martens Joost H A JHA Vermeulen Michiel M Jansen Pascal W T C PWTC Baltissen Marijke P A MPA Gorkom Britta A P Laros-van BAPL Janssen Hans H Jansen Joop H JH von Lindern Marieke M Meijer Alexander B AB van den Akker Emile E van der Reijden Bert A BA

Haematologica 20190117 7

Dominant-negative mutations in the transcription factor Growth Factor Independence-1B (GFI1B), such as GFI1B<sup>Q287*</sup>, 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. Sequestra ...[more]

PMID: 30655368

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Project description:Platelets are essential for hemostatic plug formation and thrombosis. The mechanisms of megakaryocyte (MK) differentiation and subsequent platelet production from stem cells remain only partially understood. The manufacture of megakaryocytes (MKs) and platelets from cell sources including hematopoietic stem cells and pluripotent stem cells have been highlighted for studying the platelet production mechanisms as well as for the development of new strategies for platelet transfusion. The mouse bone marrow stroma cell line OP9 has been widely used as feeder cells for the differentiation of stem cells into MK lineages. OP9 cells are reported to be pre-adipocytes. We previously reported that 3T3-L1 pre-adipocytes differentiated into MKs and platelets. In the present study, we examined whether OP9 cells differentiate into MKs and platelets using MK lineage induction (MKLI) medium previously established to generate MKs and platelets from hematopoietic stem cells, embryonic stem cells, and pre-adipocytes. OP9 cells cultured in MKLI medium had megakaryocytic features, i.e., positivity for surface markers CD41 and CD42b, polyploidy, and distinct morphology. The OP9-derived platelets had functional characteristics, providing the first evidence for the differentiation of OP9 cells into MKs and platelets. We then analyzed gene expressions of critical factors that regulate megakaryopoiesis and thrombopoiesis. The gene expressions of p45NF-E2, FOG, Fli1, GATA2, RUNX1, thrombopoietin, and c-mpl were observed during the MK differentiation. Among the observed transcription factors of MK lineages, p45NF-E2 expression was increased during differentiation. We further studied MK and platelet generation using p45NF-E2-overexpressing OP9 cells. OP9 cells transfected with p45NF-E2 had enhanced production of MKs and platelets. Our findings revealed that OP9 cells differentiated into MKs and platelets in vitro. OP9 cells have critical factors for megakaryopoiesis and thrombopoiesis, which might be involved in a mechanism of this differentiation. p45NF-E2 might also play important roles in the differentiation of OP9 cells into MK lineages cells.

Dataset Information

Molecular mechanisms of bleeding disorderassociated GFI1BQ287* mutation and its affected pathways in megakaryocytes and platelets.

Publications

Molecular mechanisms of bleeding disorderassociated GFI1B<sup>Q287*</sup> mutation and its affected pathways in megakaryocytes and platelets.

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