OC 68.3 - FLI-1 variants reduce TALIN1 expression in human megakaryocytes and platelets

Tuesday, June 25, 2024

15:15 – 15:30 ICT

Room: 211 A-D

Presenting Author(s)

ELISA GABINAUD, Master's degree

PhD student
C2VN
marseille, Provence-Alpes-Cote d'Azur, France

Co-Author(s)

LH

Laurent Hannouche

Engineering study
C2VN
MARSEILLE, Provence-Alpes-Cote d'Azur, France
MV

Mathilde Veneziano Broccia

PhD student
C2VN
MARSEILLE, Provence-Alpes-Cote d'Azur, France
Timothée Bigot

PhD
C2VN
MARSEILLE, Provence-Alpes-Cote d'Azur, France
EA

Elisa Andersen

Assistant engineer
C2VN
MARSEILLE, Provence-Alpes-Cote d'Azur, France
PS

Paul Saultier

Associate Professor
hospital APHM
MARSEILLE, Provence-Alpes-Cote d'Azur, France
Manal IBRAHIM

Pathologist
Biologic Hematology, hospital APHM, Marseille
Marseille, Provence-Alpes-Cote d'Azur, France
JS

Justine SUFFIT

Assistant engineer
C2VN
Marseille, Provence-Alpes-Cote d'Azur, France
AB

Anne Bauters

PharmD
CHU Lille, Service d'Hemostase et Transfusion - Lille FRANCE
Lille, Nord-Pas-de-Calais, France
AV

Anne Vincenot

Pathologist, PhD
CHU Paris Robert Debré, Paris, France
Paris, Ile-de-France, France
Marie-Christine ALESSI

Director
Aix Marseille Univ, Inserm 1263, inrae 1260, C2VN, APHM
Marseille, Provence-Alpes-Cote d'Azur, France
MP

Marjorie POGGI

MCU
AMU
MARSEILLE, Provence-Alpes-Cote d'Azur, France

Background: Germline variations in the FLI-1 gene, which is critical in megakaryopoiesis, have been associated with bleeding disorders, platelet aggregation defects and mild thrombocytopenia. However, the mechanisms behind these abnormalities are not fully understood.

Aims: To understand the consequences of FLI-1 variants on human megakaryocytes and platelets.

Methods: We focused on four FLI-1 variants, two of which have never been reported (Gly307Arg and Arg340Cys). Methods included immunofluorescence, western blot, luciferase reporter gene assays, siRNA gene silencing and single-cell RNAseq analysis of CD34+-derived megakaryocytes. Primary patients’s cells and cell lines were used (Meg01 and HEK293-MSR cells).

Results: Three variants (Arg337Gln, Lys345Asp, and Arg340Cys) exhibited faulty nuclear localization, two (Lys345Asp and Gly307Arg) affected protein stability whereas all showed defective transcriptional activity. 626 genes were differentially expressed in megakaryocytes, including genes associated with the "platelet activation" pathway. TLN1, was among the most downregulated genes, with 88% reduction in the amount of the TALIN1 protein in FLI-1 patients' platelets (n=5) compared to healthy controls, and 72% decrease in Meg01 cells transfected with FLI1-siRNA (n=3). ChIP-Seq data revealed FLI-1 binding regions in the TLN1 gene. Using luciferase reporter gene assays, we showed the functional role of one intronic binding region in cooperation with GATA1. All the FLI-1 variants except one (Lys345Asp) reduced this cooperative transcriptional activity.

Conclusion(s): This work describes three mechanisms that contribute to the transcriptional activity defects of the FLI-1 variants, including two new mechanisms: reduced stability of the FLI-1 protein and impaired cooperation between FLI-1 and GATA1. Defective collaborative transcription between FLI-1 variants and GATA1 can explain the deficiency of TALIN1 in FLI-1 patients' platelets, contributing to observed platelet dysfunction. Moreover, TALIN1 could serve as a potential biomarker for classifying FLI-variant pathogenicity.