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Molecular characterization of GFI1BQ287* mutation
megakaryocyte processes.4 One of the transcription fac- tors mutated in inherited bleeding and platelet disorders is Growth Factor Independence 1B (GFI1B). To date, truncat- ing mutations affecting DNA binding,5-8 missense muta- tions,9-11 and mutations that change the amount and ratio of the two naturally occurring GFI1B isoforms (p37 and p32) have been described.11,12 Bleeding tendencies may vary depending on the type of mutations, being severe for cases that express only GFI1B-p32,12 moderate to severe for dominant-negative truncating mutations,5-8 and mild to even absent for the missense mutations.9-11 Most, but not all, mutations associate with macrothrombocytopenia, a reduction in platelet a-granules, and increased CD34 expression. For some mutations, a reduction in CD42b expression, paucity of platelet δ-granules, and an increase in the numbers of morphologically abnormal megakaryo- cytes in the bone marrow have also been described. Based on these observations it can be concluded that normal GFI1B regulates a multitude of megakaryocyte-specific processes, and that the molecular mechanisms causing the abnormalities may differ.
GFI1B is a transcriptional repressor containing six C-ter- minal zinc fingers (in the case of the GFI1B-p37 isoform) and an N-terminal Snail/GFI1 (SNAG) domain, important for lysine specific demethylase (LSD1/KMD1A), REST Corepressor 1 (CoREST/RCOR1) recruitment.13 The GFI1B-p32 isoform that lacks intact zinc finger 1 and 2, also associates with LSD1-RCOR1 and is sufficient for erythropoiesis.12,14 In addition to LSD1, GFI1B recruits his- tone methyl transferases and histone deacetylases (HDAC) to target gene promoters and enhancers to induce transcriptional repression, among which GFI1B itself.15,16 Of the co-factors, LSD1 may be especially important because the majority (80%) of Gfi1b-bound regions in murine MEL cells are also enriched for Lsd1 occupancy, suggesting a strong interdependency.13 LSD1 is an essential gene in mammalian differentiation and functions, includ- ing hematopoiesis. In vitro and in vivo Lsd1 knockdown studies showed that normal granulopoiesis, erythropoiesis and megakaryopoiesis all depend on Lsd1.13,17,18
Inherited mutations in GFI1B disrupting its DNA bind- ing zinc fingers, such as GFI1BQ287*, are known to act in a dominant-negative manner.5 The GFI1B p.Q287* mutation is located within the fifth zinc finger of GFI1B and leads to a protein truncated in its DNA-binding region. As a conse- quence, the ability of GFI1BQ287* to bind GFI1B sequence motifs is disrupted.8 Importantly, its SNAG domain, cru- cial for LSD1 binding, remains intact. In this study we used the dominant-negative GFI1BQ287* mutant to unravel the mechanism by which it inhibits wildtype GFI1B and identified GFI1B regulated pathways during megakaryo- poiesis.
Methods
Mass spectrometry analysis of GFI1B-interacting proteins in MEG-01 cells
MEG-01 cells were lentivirally transduced with FUW, FUW- GFI1B-green fluorescent protein (GFP) and FUW-GFI1BQ287*-GFP and nuclear extracts were prepared as described by Dignam et al.19 Label-free GFP-pulldowns were performed using GFP-Trap beads (ChromoTek) as described by Smits et al.20 Subsequently, the pro- teins were subjected to on-bead trypsin digestion and peptides were acidified and desalted using C18-Stagetips.21 Peptides were
recorded with an LC-MS/MS Orbitrap Fusion Tribrid mass spec- trometer (ThermoFisher Scientific). Raw data were analyzed by MaxQuant (version 1.5.7.0). Proteins differentially expressed between empty vector and GFI1B or GFI1BQ287* were determined using a t-test with a false discovery rate (FDR) <0.01 and a fold change (FC) >9.2. The stoichiometry of the identified complexes was determined by dividing the iBAQ intensity in the GFI1B/GFI1BQ287* samples by the iBAQ intensity in the empty vec- tor samples.
Proliferation of GFI1B-transduced MEG-01 cells
MEG-01 cells were retrovirally transduced with pMIGR1-GFI1B variant-flag-IRES-GFP resulting in mixed cultures of GFP positive and -negative cells. The GFP% was followed using flow cytome- try for 26 days. GFP% was normalized to the starting point of the culture (day 5) using the following formula: GFP% day X/(100 – GFP% day X))/(GFP% day 5/(100 – GFP% day 5). On day 23, GFP-positive cells were FACS-sorted to determine total and endogenous GFI1B expression using quantitative real-time poly- merase chain reaction analysis.
Primary megakaryocyte cultures
CD34+ hematopoietic stem and progenitor cells were isolated from mobilized peripheral blood of healthy donors. Informed consent was given in accordance with the Declaration of Helsinki and the Dutch national and Sanquin internal ethical review boards. CD34+ cells were differentiated to megakaryocytes in modified Iscove modified Dulbecco medium (HEMAdef)22 sup- plemented with 50 ng/mL stem cell factor, 50 ng/mL throm- bopoietin, 1 ng/mL interleukin-3 and 20 ng/mL interleukin-6 for 4 days, followed by culture in 50 ng/mL thrombopoietin and 10 ng/mL interleukin-1β for 7 more days (Peprotech).23 Where indi- cated, 4 μM GSK-LSD1 (Sigma) was added subsequently and cells were cultured for an additional 2-6 days depending on the read out (2 days: expansion, CD34 and CD42b expression; 2-6 days: prolonged effect on CD42b expression and proplatelet for- mation).
Proteomics mass spectrometry analysis of platelets and megakaryocytes
Blood was taken from healthy donors and patients to generate induced pluripotent stem cells (iPSC) and isolate platelets. The study was approved by the Medical Ethical Committee of the Radboud University Medical Center, Nijmegen (2013/064) and conducted in accordance with the Declaration of Helsinki. The control iPSC line MML-6838-Cl224 and GFI1BQ287* patient iPSC lines BEL-5-Cl1 and Cl2 (Online Supplementary Figure S1) were differen- tiated towards megakaryocytes as described before.25 For label- free quantification, proteins were reduced, alkylated, trypsin- digested, desalted and concentrated. Tryptic peptides were sepa- rated by nanoscale C18 reverse phase chromatography coupled on-line to an Orbitrap Fusion Tribrid mass spectrometer (ThermoFisher Scientific) via a nanoelectrospray ion source (Nanospray Flex Ion Source, ThermoFisher Scientific). All mass spectrometry data were acquired with Xcalibur software (Thermo Scientific) and processed with the MaxQuant computational plat- form, 1.5.2.8. The RAW files, MaxQuant search results, and details about the settings are available in the PRIDE repository database26 with the dataset identifier PXD009020.
Statistical analysis
Statistical analysis for non-mass spectrometry experiments was performed in GraphPad Prism.
For more details see the Online Supplementary Methods.
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