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Molecular characterization of GFI1BQ287* mutation
teins reduced in GFI1BQ287* platelets, but was in fact mildly elevated (Online Supplementary Table S3). In addition, strongly elevated expression of CD34 was observed in GFI1BQ287* platelets, compatible with findings reported above and published reports.5,8 Other proteins enriched in GFI1BQ287* platelets were proteasomal, ribosomal, and mitochondrial proteins (Online Supplementary Figure S7; Online Supplementary Table S3). GO terms associated with downregulated proteins in GFI1BQ287* platelets were strongly related to platelet functions and a-granules, including wound healing, chemotaxis, immune response, and vesicle secretion (Figure 6D; Online Supplementary Table S2). Proteins whose levels were elevated in GFI1BQ287* platelets were particularly enriched for GO terms on mito- chondrial function and the respiratory electron transport chain (Figure 6E; Online Supplementary Table S2). In addi- tion, a multitude of other cellular processes associated with underrepresented (e.g. the Golgi and ER systems, adhesion, and the cytoskeleton) and overrepresented pro- teins (e.g. ubiquitination, RNA processing, hematopoietic differentiation, and several metabolic functions) were observed (Figure 6D,E; Online Supplementary Table S2). Thus, in addition to a major reduction in a-granule pro- teins, GFI1BQ287* platelets exhibit aberrant expression of many other proteins with various functions.
The GFI1BQ287* platelet proteome resembles that of early megakaryoblasts
As both platelets and cultured megakaryocytic cells har- boring the GFI1BQ287* mutation express the early progeni- tor marker CD34, we next asked whether GFI1BQ287* platelets also express other early megakaryocyte proteins that are normally downregulated upon terminal differen- tiation. To study this, we compared protein profiles of healthy maturing megakaryocytes with GFI1BQ287* patient-derived platelets. To this end, CD34+ cells were differentiated to megakaryocytes and harvested for mass spectrometry analysis between days 4 and 14 of differen- tiation. A total of 3,733 proteins were quantified, of which 1,668 proteins showed significantly different expression levels during megakaryocyte maturation (Figure 7A; Online Supplementary Table S4). Two main clusters of proteins showed strong upregulation (578 pro- teins) or downregulation (1,026 proteins) towards the late stages of megakaryocyte differentiation. Compatible with the role of megakaryocytes as the platelet progeni- tor cells, platelet a-granule proteins and receptors were strongly increased during megakaryocyte differentiation (Figure 7B,C). GO term analyses showed that upregulated proteins were indeed strongly related to platelet biogene- sis and function, including wound healing, (a) granules, the cytoskeleton, and cell activation (Online Supplementary Figure S8A; Online Supplementary Table S2). Downregulated proteins were associated with several GO terms related to ribosome function, RNA processing, DNA replication (including the MCM proteins) and other metabolic and biosynthetic processes (Online Supplementary Figure S8B; Online Supplementary Table S2). Next, the over- and underrepresented proteins in GFI1BQ287* platelets were compared with the protein pro- files of the maturing megakaryocytes. The majority of downregulated proteins in GFI1BQ287* platelets showed a clear pattern of upregulation in differentiating megakaryocytes (~80%) (Figure 7D), whereas the pro- teins that showed upregulation in GFI1BQ287* platelets
were generally downmodulated during megakaryocyte differentiation (~60%) (Figure 7E). As platelets represent the terminal stage of megakaryocyte maturation, our pro- teomic data show that GFI1BQ287* platelets resemble poor- ly differentiated megakaryocytes.
Discussion
The work presented here furthers our understanding on deregulated and disease-causing processes in GFI1B-related bleeding and platelet disorders. Earlier we proposed that GFI1BQ287*, observed in individuals with an inherited bleed- ing diathesis, may inhibit the function of wildtype GFI1B in a dominant-negative manner by sequestering co-repres- sors.5 Indeed, mutant GFI1BQ287* still interacts with LSD1 and its associated proteins in MEG-01 cells. Unlike GFI1B causing a decrease in proliferative capacity, forced expres- sion of GFI1BQ287* in MEG-01 cells resulted in increased pro- liferation. This increase was nullified upon introduction of single point mutations in GFI1BQ287* which abrogate the interaction with LSD1. In line with these findings we observed that disruption of the GFI1B-LSD1 interaction, using GSK-LSD1, induces GFI1BQ287*-like phenotypes in CD34+ cell-derived megakaryocytes, including the down- regulation of CD42b. Yet, GFI1BQ287* iPSC-derived megakaryocytes did not show impaired CD42b expres- sion. This could be explained by variation in CD42b expression observed in affected individuals, with highest levels being comparable to those observed in non-affected individuals.5 Alternatively, this may be explained by differ- ences in in vivo and in vitro differentiation cues. Nevertheless, we propose that GFI1BQ287* inhibits wildtype GFI1B by inhibiting LSD1-dependent processes through competitive sequestration of LSD1 and its binding part- ners.
The relevance of the GFI1B-LSD1 interaction in megakaryoblast expansion is substantiated by observa- tions in mice following in vivo Lsd1 silencing. This resulted in Meis1 overexpression and increased megakaryocyte numbers in the bone marrow as well. Of note, here we found MEIS1 only in GFI1BQ287* iPSC-derived megakaryo- cytic cells but not in control iPSC-derived cells, indicating elevated expression in the former. Interestingly, Lsd1- silenced mice also exhibited megakaryocyte abnormali- ties, thrombocytopenia and a reduction of platelet gran- ules, similar to the phenotypes of patients harboring the dominant-negative GFI1BQ287* mutation.18 This could mean that the functional interplay between GFI1B and LSD1 is not limited to controlling megakaryoblast proliferation and differentiation, but that it is also required for terminal maturation and the generation of granules and platelets. In addition, this interplay is not restricted to megakary- opoiesis, but has also been shown to be essential for the transition of endothelial to hematopoietic stem cells and erythrocyte development.13,14,34
In addition to GFI1BQ287*, two other truncating GFI1B mutations that disrupt the DNA binding domain have been identified, G272fs8 and H294fs,6 both of which lead to a similar bleeding and platelet disorder. These GFI1B mutations also act in a dominant-negative manner. Thus, LSD1 sequestration by mutant GFI1B that cannot bind its transcriptional targets may be a general mechanism through which a collection of GFI1B mutations acts. In these cases, the mutation is present in both GFI1B iso-
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