Identification of Vascular and Hematopoietic Genes Downstream of etsrp by Deep Sequencing in Zebrafish

The transcription factor etsrp/Er71/Etv2 is a master control gene for vasculogenesis in all species studied to date. It is also required for hematopoiesis in zebrafish and mice. Several novel genes expressed in vasculature have been identified through transcriptional profiling of zebrafish embryos overexpressing etsrp by microarrays. Here we re-examined this transcriptional profile by Illumina RNA-sequencing technology, revealing a substantially increased number of candidate genes regulated by etsrp. Expression studies of 50 selected candidate genes from this dataset resulted in the identification of 39 new genes that are expressed in vascular cells. Regulation of these genes by etsrp was confirmed by their ectopic induction in etsrp overexpressing and decreased expression in etsrp deficient embryos. Our studies demonstrate the effectiveness of the RNA-sequencing technology to identify biologically relevant genes in zebrfish and produced a comprehensive profile of genes previously unexplored in vascular endothelial cell biology

Dysregulation of PLDN (pallidin) is a mechanism for platelet dense granule deficiency in RUNX1 haplodeficiency.

BACKGROUND: Inherited RUNX1 haplodeficiency is associated with thrombocytopenia and platelet dysfunction. Dense granule (DG) deficiency is reported in patients with RUNX1 haplodeficiency, but the molecular mechanisms are unknown. Platelet mRNA expression profiling in a patient previously reported by us with a RUNX1 mutation and platelet dysfunction showed decreased expression of PLDN (BLOC1S6), which encodes for pallidin, a subunit of BLOC-1 (biogenesis of lysosome-related organelles complex-1) involved in granule biogenesis. PLDN mutations in the pallid mouse and the Hermansky-Pudlak syndrome (HPS)-9 are associated with platelet DG deficiency. OBJECTIVES: We postulated that PLDN is a RUNX1 target and that its decreased expression leads to platelet DG deficiency in RUNX1 haplodeficiency. RESULTS: Platelet pallidin and DG were decreased in our patient. This was also observed in two siblings from a different family with a RUNX1 mutation. Chromatin immunoprecipitation and electrophoretic mobility shift assays using phorbol ester-treated human erythroleukemia (HEL) cells showed RUNX1 binding to RUNX1 consensus sites in the PLDN1 5′ upstream region. In luciferase reporter studies, mutation of RUNX1 sites in the PLDN promoter reduced activity. RUNX1 over-expression enhanced and RUNX1 downregulation decreased PLDN1 promoter activity and protein expression. RUNX1 downregulation resulted in impaired handling of mepacrine and mislocalization of DG marker CD63 in HEL cells, indicating impaired DG formation, with recapitulation of findings on PLDN downregulation. CONCLUSIONS: These studies provide the first evidence that PLDN is a direct target of RUNX1 and its dysregulation is a mechanism for platelet DG deficiency associated with RUNX1 haplodeficiency