FLI1 and GATA1 govern TLN1 transcription: new insights into FLI1-related platelet disorders

Abstract

Germline variants of FLI1, essential for megakaryopoiesis, are linked to bleeding disorders, platelet aggregation defects and mild thrombocytopenia. However, the mechanisms behind these abnormalities remain unclear. This study aims to elucidate the impact of FLI1 variants on human megakaryocytes and platelets. We focused on four FLI1 variants, two of which are novel: p.G307R and p.R340C. We assessed the impact of FLI1 variants on megakaryopoiesis using single-cell RNA sequencing, and defects were confirmed in patients' platelets and cell lines. Results showed variants p.R337Q, p.K345E and p.R340C exhibited faulty nuclear localization and defective transcriptional activity in vitro and variants p.K345E and p.G307R affected protein stability. A total of 626 genes were differentially expressed in patient megakaryocytes, including genes associated with the platelet activation pathway. TLN1 was among the most down-regulated genes, with an 88% reduction in talin-1 protein levels in FLI1 patients' platelets. Analysis of chromatin immunoprecipitation sequencing data revealed FLI1-binding regions in the TLN1 gene. Luciferase reporter gene assays revealed the functional role of an intronic binding region in cooperation with GATA1. FLI1 variants were linked to reduced cooperative transcriptional activity. These findings reveal novel mechanisms underlying the pathogenicity of FLI1 variants. Defective cooperation between FLI1 variants and GATA1 may play a role in talin-1 deficiency in FLI1 patients' platelets, thus contributing to platelet dysfunction. Moreover, talin-1 could serve as a biomarker for classifying the pathogenicity of FLI1 variants.

Figure 1.

Functional characterization of the two novel FLI1 variants. (A) Schematic diagram of the FLI1 protein. The functional N-terminal Pointed (PNT) and C-terminal ETS (ETS) DNA-binding domains are depicted. The position of the variations in FLI1 is indicated in red (new variations described in this study) or black (previously reported variations). (B) MSR cells were co-transfected with an empty vector, wild-type (WT) FLI1 or FLI1 variant constructs, including the previously reported FLI1 variants, along with the Firefly luciferase reporter plasmid containing three tandem copies of the ETS-binding site upstream of the HSV TK promoter (E743tk80Luc) and the pGL4.73 Renilla luciferase control vector. The Firefly to Renilla luminescence ratios (Fluc/Rluc) were calculated to normalize for transfection efficiency and are expressed as percentage relative to the empty vector. Data represent mean ± standard error of the mean (SEM) of ten independent experiments; **P<0.01, ***P<0.001, ****P<0.0001 versus WT (Kruskal-Wallis test). (C) Representative Western blot analysis of FLI1 expression in MSR cells transfected with WT FLI1 or FLI1 variant constructs. We verified FLI1 expression using an anti-HA antibody. GAPDH was used as a protein loading control. (Bottom) Results of the densitometric analysis are expressed as mean ± SEM. Three independent experiments were performed. (D) Representative Western blot analysis of MYH10 expression in washed platelets from FLI1 variant carriers. R340C was compared to two unaffected family members (father and mother) and one unrelated control. G307R was compared to two unrelated controls. GAPDH was used as a protein loading control. (Right) Densitometric analysis results were normalized to GAPDH and expressed as mean ± SEM. (E) Representative immunofluorescence microscopy images of H9C2 cells transfected with WT FLI1 or FLI1 variant constructs visualized using immunofluorescence after FLI1 (anti-HA antibody), nucleus (DAPI) and ACTIN (rhodamine phalloidin) staining; scale bar, 5 µm. (F) Effect of FLI1 variations on the half-life of FLI1 protein. MSR cells were transfected with WT FLI1 or FLI1 variant constructs. After 48 hours (hr), cells were treated with cycloheximide (CHX) for the indicated times (0, 5.5 or 9 hr). Cells were lysed, and cell lysates were then subjected to Western blot analysis. (Right) Densitometric analysis results are expressed as mean ± SEM. FLI1 levels at each time point are represented relative to the initial levels at time zero (t0). Two independent experiments were performed.

Figure 2.

Structural model of FLI1 interactions. (A) Structure of FLI1 bound to double-stranded DNA (PDB: 5e8i). FLI1 shown in pale green and the double-stranded DNA in pink cartoon. FLI1 residues G307, R337, R340, and K345 are colored in orange. Nucleotides, R337, R340 (Inset A), and K345 (Inset B) are shown in sticks at the FLI1/DNA interface. Hydrogen bonds and salt bridges are depicted in black dashed lines (< 3.5Å). Oxygen atoms are in red, nitrogen in blue, and phosphorus in orange. (B) Structure of FLI1 dimer (PDB: 5e8i). FLI1 monomers 1 and 2 shown in pale green and cyan cartoon, respectively, with residues G307, R337, R340, and K345 colored in orange. Inset A shows the FLI1 1/2 dimer with residues G307 and contact residues N306, D361, and F360 in sticks. Atoms and contacts are represented as in (A).

Figure 3.

Single-cell RNA sequencing of cells derived from the FLI1 R337Q variant carrier and healthy controls. (A) UMAP plot of the FLI1 R337Q variant and control cells colored according to identified hematopoietic cell types: hematopoietic stem/progenitor cells (HSPC), common myeloid progenitors (CMP), megakaryocyte (MK)-primed CMP, granulocyte-monocyte progenitors (GMP), MK-erythroid progenitors (MEP), and progenitor and mature MK (MKP/MK). (B) Violin plots showing FLI1 mRNA levels in different hematopoietic cell types from healthy subjects (left) and the FLI1 patient (right). Each cell type is color-coded. (C) Bubble plot depicting the top down-regulated enriched KEGG biological pathways based on differentially expressed genes (DEG) by cell type (classified by P value). Dot sizes reflect the ratio of genes enriched in this pathway to the total number of genes in the pathway. Bubbles are color-coded according to cell type (HSPC, CMP, MK-primed CMP, GMP, MEP, MKP/Mk). (D) Bar plot illustrating the fold change of DEG in the platelet activation pathway, specifically of the MKP-MK cell type. (E) I-cis target results on the 24 genes of the platelet activation pathway. PWM: position weight matrix; NES: normalized enrichment score.

Figure 4.

Talin-1 levels and fibrinogen binding in FLI1-deficient cells. (A) Western blot analysis of talin-1 expression in washed platelets from controls and FLI1 variant carriers. GAPDH was used as a protein loading control. Each patient was compared with age- and sex-matched control subjects. For each patient and matched controls, blood samples were collected at the same time and processed in parallel to avoid any bias. Quantification of band intensity is shown on the right. ***P<0.001 (Mann-Whitney test). (B) Binding of labeled fibrinogen (Fibrinogen-FITC) to washed platelets from controls and FLI1 variant carriers (K345E▪ and R340C▲) upon activation with 10 μM TRAP6 for 15 minutes. Fibrinogen binding fold change values represent the mean fluorescent intensity (MFI) of fibrinogen bound by stimulated platelets, subtracted from the MFI under resting conditions. (C) FLI1 siRNA were transfected into MEG-01 cells. Endogenous FLI1 and talin-1 expression levels were quantified via Western blot analysis (N=3 per group). GAPDH was used as a protein loading control. (Right) Band intensity was quantified.

Figure 5.

Identification and functional analysis of FLI1 regulatory regions in the TLN1 gene. (A) Visualization of ChIP-seq peaks in the TLN1 gene. FLI1, SCL, RUNX1 and GATA1 binding loci in the TLN1 promoter region (purple) and intron 1 (yellow, orange, and green) are shown. BS: binding site. (B) Luciferase activity in HEL cells transfected with various pGL3 luciferase reporter vectors, including the empty vector (gray), the TLN1 promoter (purple), or TLN1 intron 1 regions (binding site 1 in yellow, binding site 2 in orange, and binding site 3 in green). The dual-luciferase assay involved sequential measurement of Firefly and Renilla luciferase activity in the same sample; results are presented as the ratio of Firefly to Renilla activity (Fluc/Rluc). The pGL3 vector lacking regulatory regions (empty vector) served as a normalization control. Each plasmid was assayed in three independent transfection experiments. *P<0.05 versus pGL3-empty (Mann-Whitney test). (C) Luciferase activity in MSR cells transfected with empty luciferase reporter vector (pGL3 empty) or luciferase vector containing the intronic binding site 3 of TLN1 (intronic BS3), along with the pCDNA-FLI1, pCDNA-GATA1, and pCDNA-SCL vectors as specified. The pCDNA empty vector (lacking transcription factors [TF]) served as a normalization control. Each plasmid was assayed in 3-6 separate transfection experiments. *P<0.05, **P<0.01, ***P<0.001 versus pCDNA-empty vector (Kruskal-Wallis test). ^##P<0.01 (Mann-Whitney test). (D) Luciferase activity in MSR cells transfected with intronic BS3 pGL3 luciferase reporter vector and pCDNA-GATA1 with WT FLI1 or FLI1 variants as indicated. Each plasmid was assayed in 2-5 separate transfection experiments. *P<0.05 versus WT FLI1 + GATA1 (Mann-Whitney test).

Figure 6.

FLI1 and GATA1 cooperation and their interaction with TLN1 intronic binding site 3. (A) Assessment of the impact of FLI1 and GATA1 overexpression on talin-1 protein levels. (Top) Western blot analysis of talin-1, FLI1 (anti-FLI1 antibody), and GATA1 (anti-GATA1 antibody), in MEG-01 lysates, either non-transduced (NT) or transduced with FLI1, GATA1 or both (14 days post transduction). GAPDH served as the loading control. (Bottom) Densitometric analysis results were normalized to GAPDH and are expressed as the mean ± standard error of the mean (SEM) of three independent experiments. *P<0.05 versus NT (Kruskal-Wallis test). (B) EMSA assay performed with biotin-labeled TLN1 intronic binding site 3 DNA probes and nuclear protein extracts from MSR cells transfected with either an empty vector, FLI1, or GATA1. Conditions 2 to 4 used 400 ng of nuclear extracts from cells transfected with the empty vector, FLI1, or GATA1, respectively. Conditions 5 to 7 used 400 ng of nuclear extracts from cells transfected with FLI1, combined with increasing amounts of nuclear extracts from cells transfected with GATA1 (120, 240, and 400 ng). Wedge symbol denotes those increasing GATA1 concentrations. No shift was observed in reactions without protein extract (condition 1) or with extracts from cells transfected with the empty vector (condition 2). A shift band was detected in reactions using extracts from FLI1-transfected (condition 3) or GATA1-transfected cells (condition 4), as indicated by the arrows. In reactions with extracts from cells transfected with both FLI1 and GATA1 (conditions 5 to 7), a higher molecular weight band shift was observed. (C) The physical interactions between WT FLI1 or FLI1 variants and GATA1 in MSR cells were assessed using a proximity ligation assay, as described in the Methods. Protein complexes of interest were visualized as red fluorescent dots, with DAPI-stained nuclei depicted in blue; scale bar, 5 µm. (Top row) Microscopy images at 40X magnification. (Bottom row) Zoomed-in view of a specific area of the top row images (indicated by a white dashed square). Quantification of the mean fluorescence intensity of red dots per transfected cell nucleus is shown at the bottom. *P<0.05, ***P<0.001 (Mann-Whitney test). FD: FLI1/DNA complexes; GD: GATA1/DNA complexes; GFD: FLI1, GATA1, and DNA complexes.