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R. van Oorschot et al.
forms and they will inhibit wildtype GFI1B-p37 and GFI1B-p32. However, because the expression of GFI1B- p32 is much lower than that of GFI1B-p37,14 LSD1 seques- tration by GFI1BQ287*-p32 will be less than that by GFI1BQ287*-p37. The molecular mechanisms causing megakaryocyte and platelet defects for other reported
AB
GFI1B mutations are likely different because the DNA binding and LSD1-interacting domains remain intact in these mutants.
We observed that GFI1B mainly associates with the LSD1-RCOR-HDAC repressor complex (also called BRAF- HDAC complex in neural development) containing LSD1,
D
C
E
Figure 7. The GFI1BQ287* platelet proteome reveals impaired megakaryocyte differentiation. The proteome of in vitro-differentiating megakaryocytes (MK) was ana- lyzed by label-free mass spectrometry (see Methods). Differentially expressed proteins were determined using analysis of variance (false discovery rate <0.05, s0=0.5). (A) Heat-map with hierarchical clustering of the 1,668 proteins showing significantly different expression between any of the analyzed days of MK differen- tiation, i.e. day 4, 5, 6, 7, 8, 9, 10, 11, and 14 of differentiation (from left to right) with three replicates per day of culture. Relative protein levels [z-scored log2 label- free quantification (LFQ)] are shown with imputed values in the case a protein was not detected. (B) Expression levels in cultured MK of platelet a-granule proteins during MK differentiation. (C) Expression levels in cultured MK of platelet receptors during MK differentiation. (D-E) Relative expression levels in cultured MK of pro- teins with significantly increased or decreased levels in GFI1BQ287* platelets: 272 of the 395 downregulated proteins (D) and 503 of the 610 upregulated proteins (E) in GFI1BQ287* platelets were identified in the MK. Magenta/blue shades in the heat-maps correspond to decreased expression levels and yellow/orange shades to increased expression levels; a gray color indicates that a protein was not identified in a given MK sample (Panels D and E only). THBS1: thrombospondin-1; VWF: von Willebrand factor; PF4: platelet factor 4; ANGPT1: angiopoietin-1; SELP: selectin P; ITGA2B: integrin subunit alpha 2b; ITGB: integrin subunit beta; CD36: cluster of differentiation 36; GP1BA: glycoprotein Ib platelet subunit alpha; GP9: glycoprotein 9.
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haematologica | 2019; 104(7)