- Anouck Wijgaerts1,
- Christine Wittevrongel1,
- Chantal Thys1,
- Timothy Devos2,
- Kathelijne Peerlinck1,
- Marloes R. Tijssen3,
- Chris Van Geet2 and
- Kathleen Freson1,*
- 1 Dept. Cardiovascular Sciences, Center for Molecular and Vascular Biology, KU Leuven, Leuven, Belgium;
- 2 Department of Haematology, University Hospitals Leuven, Leuven, Belgium;
- 3 NHS Blood and Transplant, Cambridge Biomedical Campus, Cambridge, United Kingdom
- ↵* Corresponding author; email:
Gray platelet syndrome is named after the gray appearance of platelets due to absence of alpha(α)-granules. It is caused by recessive mutations in NBEAL2, resulting in macrothrombocytopenia and myelofibrosis. Though using the term gray platelets for GATA1 deficiency has been debated, a reduced number of α-granules has been described for macrothrombocytopenia due to GATA1 mutations. We compared platelet size and number of α-granules for two NBEAL2 and two GATA1 deficient patients and found reduced numbers of α-granules for all, with the defect being more pronounced for NBEAL2 deficiency. We further hypothesized that the granule defect for GATA1 is due to a defective control of NBEAL2 expression. Remarkably, platelets from two patients and GATA1-deficient mice express almost no NBEAL2. The differentiation of GATA1 patient-derived CD34+ stem cells to megakaryocytes showed defective proplatelet and α-granule formation with strongly reduced NBEAL2 protein and RNA expression. ChIP sequencing revealed 5 GATA binding sites in a regulatory region 31 kb upstream of NBEAL2 covered by an H3K4Me1 mark indicative of an enhancer locus. Luciferase reporter constructs containing this region confirmed its enhancer activity in K562 cells and mutagenesis of the GATA1 binding sites resulted in significantly reduced enhancer activity. Moreover, DNA binding studies showed that GATA1 and GATA2 physically interact with this enhancer region. GATA1 depletion using siRNA in K562 cells also resulted in reduced NBEAL2 expression. In conclusion, we here show a long-distance regulatory region with GATA1 binding sites as strong enhancer for NBEAL2 expression.
- Received July 16, 2016.
- Accepted January 10, 2017.
- Copyright © 2017, Ferrata Storti Foundation