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 patient 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 downregulated genes, with an 88% reduction in talin-1 protein levels in FLI1 patient 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 patient platelets, thus contributing to platelet dysfunction. Moreover, talin-1 could serve as a biomarker for classifying the pathogenicity of FLI1 variants.