OC 78.5 - G6b-B controls transcription factor expression during megakaryocyte maturation

Background: Platelets are produced by megakaryocytes (MKs), which differentiate from hematopoietic stem cells in the bone marrow. Various transcription factors, including GATA1, RUNX1 and FLI1, are crucial for MK development. Despite their importance, the precise mechanisms governing the induction and repression of transcription factors during MK differentiation remain poorly understood. Furthermore, G6b-B, an immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor with an MK lineage-specific expression pattern, harbors an enigmatic function. Notably, both patients and mice lacking G6b-B expression exhibit macrothrombocytopenia and myelofibrosis. We recently identified G6b-B as a key player in megakaryocyte maturation (Becker, Nagy et al. Blood Adv. 2022) however, the molecular mechanisms underlying this role remain elusive.

Aims: To identify the role of G6b-B in regulating the MK transcriptional program.

Methods: Mice lacking G6b-B expression (Mpig6bmut) were compared to wild-type mice by analyzing native bone marrow cells. MKs were enriched using magnetic cell separation and processed for single-cell RNA sequencing (scRNA-seq) utilizing the 10x Chromium system. Data was analysed using CellBender and the Seurat package. MK development was assessed using flow cytometry and immunofluorescence microscopy.

Results: Sequencing of about 40,000 cells/genotype allowed for high-resolution analysis of MKs and their heterogeneity. Our data identified that loss of functional G6b-B leads to aberrant MK subtype distribution including substantial expansion of immature MKs and immune-modulatory MKs, accompanied by a decrease in the proportion of platelet-generating mature MKs. In mature MKs, the expression of several MK-specific transcripts was substantially reduced. Remarkably, gene expression analysis demonstrated that approximately 10% of the genes exhibited increased expression in mutant MKs were transcription factors.

Conclusion(s): Loss of G6b-B profoundly impacts MK development, marked by an altered MK subtype distribution. Our findings underscore the critical influence of G6b-B on the transcriptional program that governs MK maturation, highlighting its novel capacity to orchestrate the expression and repression of transcription factors.