Defective Rab1b Related Megakaryocytic ER-to-Golgi Transport in RUNX1 Haplodeficiency

RUNX1, a major hematopoietic transcription factor, plays a critical role in megakaryopoiesis, megakaryocytic (MK) maturation and platelet production. Patients with RUNX1 haplodeficiency have thrombocytopenia, abnormal platelet function and impaired granule secretion. Defective dense and/or -alpha granules are a major finding in these patients. We have reported a patient with inherited thrombocytopenia, decreased granules, and abnormal platelet function (Gabbeta et al , Blood 87: 1368-76, 1996) with a heterozygous RUNX1 mutation (c.508+1G>1) (Sun et al , Blood 103: 948-54, 2004). Platelet expression profiling revealed down-regulation of several genes including ALOX12 , MYL9 , PRKCQ , PF4, and PLDN (Sun et al J Thromb Haemost 5: 146-154, 2007) and we have shown these to be direct RUNX1 transcriptional targets. In addition, a small G protein, RAB1B, was significantly decreased on expression profiling of the patient platelets relative to control subjects (fold change: 0.32, p RAB1B mRNA was decreased by 50% compared to healthy 5 subjects. Immunofluorescence studies showed that Rab1b protein was decreased in patient platelets compared to that of normal subjects. RAB GTPases are key players in vesicle trafficking, granule targeting of proteins, and secretion. Rab1b has been implicated in ER-to-Golgi transport in COS cells, but cell biological roles of RAB1B in MK cells are unexplored. We addressed the hypothesis that RAB1B is a transcriptional target of RUNX1 and regulates ER-to-Golgi transport in MK cells. In silico analysis of the RAB1B promoter region (-1/-785 bp from the ATG) revealed 4 RUNX1 consensus sites: site-1 (-440/-435), site-2 (-622/-617), site-3 (-660/-655) and site-4 (-770/-765). Following phorbol 12-myristate 13-acetate treatment of human erythroleukemia (HEL) cells to induce MK transformation, chromatin immunoprecipitation (ChIP) revealed RUNX1 binding to RAB1B sites 1 and 4 but not to other sites. Electrophoretic mobility shift assays using HEL cell proteins showed RUNX1 binding to these sites. In luciferase reporter assays, mutation individually of sites 1 and 4 reduced the promoter activity by 90%, compared to that of full-length promoter. RUNX1 over-expression enhanced RAB1B promoter activity by 2-fold and RAB1B protein by ~30%. Enhancement of promoter activity was abrogated by mutations of sites 1 and 4, indicating that these sites were functional. RUNX1 siRNA reduced RAB1B promoter activity and RAB1B protein. We studied vesicle trafficking from ER-to-Golgi in HEL cells using vesicular stomatitis virus G envelope glycoprotein (VSVG) tagged with enhanced green fluorescence protein (EGFP) and GalT on siRNA downregulation of RUNX1 or RAB1B . (Presley JF et al, Nature 389: 81-85, 1997). VSVG-GFP colocalized with GalT in the Golgi in control siRNA-transfected cells, whereas in RUNX1 knockdown cells, VSVG was not colocalized with GalT; moreover, Golgi structures were disrupted. Similar results were obtained in RAB1B downregulation by siRNA. These effects of RUNX1 knockdown were reversed by RAB1B overexpression. Together these results suggest that RAB1B downregulation may be the primary cause of the impaired ER-to-Golgi transport by RUNX1 downregulation. Conclusions: These studies show that RAB1B is a key regulator of vesicle trafficking in the secretory and endocytic pathway in megakaryocytes and platelets. This is the first evidence that RAB1B is a direct transcriptional target of RUNX1 and provides a mechanism for RAB1B down regulation in our RUNX1 haplodeficient patient. One of the hallmarks of RUNX1 haplodeficiency is defective granule formation and secretion. We propose RAB1B reduction of ER-to-Golgi transport contributes to defects in granule formation and secretion in patients with RUNX1 deficiency. Disclosures No relevant conflicts of interest to declare.