title: The transcription factor GATA1 regulates NBEAL2 expression through a long-distance enhancer.
abstract: Gray platelet syndrome is named after the gray appearance of platelets due to the absence of α-granules. It is caused by recessive mutations inNBEAL2, 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 toGATA1mutations. 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, expressed 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 ribonucleic acid expression. Chromatin immunoprecipitation sequencing revealed 5 GATA binding sites in a regulatory region 31 kb upstream ofNBEAL2covered by a 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 small interfering ribonucleic acid in K562 cells also resulted in reduced NBEAL2 expression. In conclusion, we herein show a long-distance regulatory region with GATA1 binding sites as being a strong enhancer for NBEAL2 expression.
authors.affiliation: Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, KULeuven, Belgium.
authors.forename: Anouck
authors.initials: A
authors.lastname: Wijgaerts
authors.affiliation: Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, KULeuven, Belgium.
authors.forename: Christine
authors.initials: C
authors.lastname: Wittevrongel
authors.affiliation: Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, KULeuven, Belgium.
authors.forename: Chantal
authors.initials: C
authors.lastname: Thys
authors.affiliation: Department of Haematology, University Hospitals Leuven, Belgium.
authors.forename: Timothy
authors.initials: T
authors.lastname: Devos
authors.affiliation: Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, KULeuven, Belgium.
authors.forename: Kathelijne
authors.initials: K
authors.lastname: Peerlinck
authors.affiliation: NHS Blood and Transplant, Cambridge Biomedical Campus, UK.|Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, UK.
authors.forename: Marloes R
authors.initials: MR
authors.lastname: Tijssen
authors.affiliation: Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, KULeuven, Belgium.|Department of Pediatrics, University Hospitals Leuven, Belgium.
authors.forename: Chris
authors.initials: C
authors.lastname: Van Geet
authors.affiliation: Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, KULeuven, Belgium kathleen.freson@med.kuleuven.be.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Freson
mesh_terms: D000483:Alleles; D001792:Blood Platelets / Q000378:metabolism; D001798:Blood Proteins / Q000235:genetics*; D002454:Cell Differentiation / Q000235:genetics; D004742:Enhancer Elements, Genetic; D050982:GATA1 Transcription Factor / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005786:Gene Expression Regulation; D005808:Genes, Recessive; D017930:Genes, Reporter; D050172:Genes, X-Linked; D056726:Genetic Association Studies; D020022:Genetic Predisposition to Disease; D006720:Homozygote; D006801:Humans; D008533:Megakaryocytes / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D013921:Thrombocytopenia / Q000097:blood / Q000235:genetics / Q000473:pathology
chemical_list: D001798:Blood Proteins; D050982:GATA1 Transcription Factor; C561485:NBEAL2 protein, human; D034741:RNA, Small Interfering