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R. van Oorschot et al.
Results
GFI1B and GFI1BQ287* interact with the LSD1-RCOR1-HDAC1/2 complex
Several GFI1B variants have been identified in inherited bleeding and platelet disorders.5,6,8,9 The majority of the pathogenic variants, such as GFI1B p.Q287*, result in a truncation disrupting the DNA binding capacity. Truncated GFI1B inhibits the wildtype protein in a domi- nant-negative manner.5,8 With the SNAG domain intact, we hypothesized that GFI1BQ287* can compete with GFI1B for the recruitment of epigenetic modifiers. To test this hypothesis, we first identified proteins interacting with GFI1B and GFI1BQ287*. To this end, C-terminally GFP- tagged GFI1B and GFI1BQ287* were expressed in megakaryoblastic MEG-01 cells. GFP-pulldown followed by on-bead trypsin digestion and mass spectrometry analysis identified the following GFI1B interacting pro- teins: LSD1, RCOR1/3, HDAC1/2, GSE1, HMG20B, PHF21A, and GFI1 (Figure 1A). Of the detected GFI1B interactors, LSD1 was most abundant, together with RCOR1/3 and HDAC1/2 (Figure 1B). The proteins GSE1, HMG20B, and PHF21A are all known to be subunits of the LSD1-RCOR-HDAC complex, also known as the CoREST (RCOR1) complex.27 The identified interactome of GFI1BQ287* was strongly similar to that of GFI1B, with ZMYM3 as the only significant differential interactor (Figure 1C; Online Supplementary Figure S2), indicating that the DNA-binding defective GFI1BQ287* mutant recruits vir- tually the same co-factors as GFI1B.
GFI1BQ287* functions in a dominant-negative manner by sequestering LSD1
The GFI1B p.Q287* mutation results in an increase in megakaryocyte expansion in vivo and in vitro.5 To investi- gate whether sequestering of LSD1 by GFI1BQ287* is impor- tant for this effect, we introduced a P2A or K8A mutation in the SNAG domain, previously described to disrupt effi- cient LSD1 recruitment to GFI1 and GFI1B.13,28 We con- firmed impaired LSD1 recruitment in GFI1BP2A/K8A-GFP transduced MEG-01 cells using GFP-trap bead-mediated pulldown (Figure 1D). Furthermore, GFI1BQ287*, GFI1BP2A and GFI1BK8A failed to inhibit endogenous GFI1B expres- sion in MEG-01 cells, in contrast to wildtype GFI1B (Figure 1E-F). Next, we used MEG-01 as a megakaryoblast model to study the effect of GFI1BQ287* on cell expansion. In expansion-competition cultures containing transduced and non-transduced cells, GFI1B-overexpressing cells were rapidly overgrown by non-transduced cells, while the opposite was observed following expression of dominant- negative GFI1BQ287* (Figure 2A). Thus, forced GFI1B expres- sion inhibits MEG-01 expansion and dominant-negative GFI1BQ287* results in enhanced expansion, the latter being in line with observations in GFI1B p.Q287* affected indi- viduals.
To determine whether the interaction with LSD1 is important for the inhibitory effect on expansion of GFI1B, we separately introduced the P2A and K8A mutations in GFI1B. Expression of GFI1BP2A or GFI1BK8A nullified the inhibitory effect of GFI1B on expansion (Figure 2B,C; Online Supplementary Figure S3). Thus, an intact LSD1- interacting SNAG domain is required for both inhibition of MEG-01 expansion and repression of the endogenous GFI1B locus.
To investigate whether the LSD1 interaction is relevant
for the dominant-negative effect in MEG-01, we intro- duced the P2A and K8A mutations into GFI1BQ287*. This showed that similar levels of ectopic GFI1BP2A+Q287* and GFI1BK8A+Q287* expression did not affect MEG-01 expansion, in contrast to GFI1BQ287* (Figure 2D,E; Online Supplementary Figure S3). Together, these data indicate that GFI1B limits MEG-01 expansion through the LSD1-interacting SNAG domain. GFI1BQ287* may affect this function in a dominant- negative manner by sequestering LSD1.
Chemical disruption of the GFI1B-LSD1 interaction in normal megakaryocytes results in abnormalities seen in GFI1B p.Q287* mutated megakaryocytes
LSD1 sequestering by GFI1BQ287* in MEG-01 resulted in enhanced proliferation. To determine whether LSD1 sequestration is relevant for other megakaryocyte abnor- malities, we inhibited the GFI1B-LSD1 interaction during megakaryocyte differentiation by treating CD34+ cell- derived megakaryoblasts with the small molecule GSK- LSD1. This inhibitor binds covalently to the LSD1 cofac- tor FAD, and thereby sterically hinders binding of GFI1B.29 We confirmed the disrupted LSD1-GFI1B binding by the absence of co-precipitated LSD1 upon GFP-trap bead- mediated pulldown in the presence of GSK-LSD1 (Figure 3A). To study effects on megakaryopoiesis, CD34+ cells were differentiated towards megakaryocytes in the pres- ence of GSK-LSD1 for 48 h. Quantification of cell surface marker expression showed sustained CD34 expression and impaired CD42b expression compared to the expres- sion in mock-treated cells (Figure 3B-D). Besides this, a 2- fold increase in megakaryoblast expansion was observed in the presence of GSK-LSD1 (Figure 3E). CD42b expres- sion remained low after 3-6 days of exposure to GSK- LSD1 (Online Supplementary Figure S4C) and we observed that these cells were unable to form proplatelets after 6 days (Figure 3F, Online Supplementary Figure S4). These phenotypes are in line with observations in individuals with the GFI1B p.Q287* mutation.5 Together with data presented in the previous section, this strongly suggests that mutant GFI1BQ287* functions in a dominant-negative manner by sequestering LSD1, resulting in developmental megakaryocyte abnormalities.
GFI1BQ287* induced pluripotent stem cell-derived megakaryocytic cells show disturbed megakaryocyte differentiation
The morphological megakaryocyte abnormalities, their increased expansion, and sustained CD34 expression indi- cate that megakaryocyte differentiation is severely dis- turbed in GFI1BQ287*-mutated individuals. As the availabili- ty of CD34+ progenitors and megakaryocytes from patients is limited, we generated iPSC lines from a control individual (MML-6838-Cl2)24 and an individual harboring the GFI1BQ287* mutation (BEL-5-Cl1/2) to study megakaryopoiesis in more detail (Online Supplementary Figure S1). iPSC colonies were differentiated towards megakaryocytes using a two-dimensional differentiation protocol.25 May-Grünwald Giemsa-stained cytospins and flow cytometry revealed enlarged cells with sometimes polyploidization for control megakaryocytic cells, with co-expression of CD41a and CD42b confirming megakaryocyte differentiation (Figure 4A,B). In contrast, differentiation of GFI1BQ287* iPSC resulted in a homoge- neous appearance of small, pale cells (Figure 4A). In addi- tion, GFI1BQ287* iPSC-derived megakaryocytic cells
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haematologica | 2019; 104(7)