title: Sp1/Sp3 transcription factors regulate hallmarks of megakaryocyte maturation and platelet formation and function.
abstract: Sp1 and Sp3 belong to the specificity proteins (Sp)/Krüppel-like transcription factor family. They are closely related, ubiquitously expressed, and recognize G-rich DNA motifs. They are thought to regulate generic processes such as cell-cycle and growth control, metabolic pathways, and apoptosis. Ablation of Sp1 or Sp3 in mice is lethal, and combined haploinsufficiency results in hematopoietic defects during the fetal stages. Here, we show that in adult mice, conditional pan-hematopoietic (Mx1-Cre) ablation of either Sp1 or Sp3 has minimal impact on hematopoiesis, whereas the simultaneous loss of Sp1 and Sp3 results in severe macrothrombocytopenia. This occurs in a cell-autonomous manner as shown by megakaryocyte-specific (Pf4-Cre) double-knockout mice. We employed flow cytometry, cell culture, and electron microscopy and show that although megakaryocyte numbers are normal in bone marrow and spleen, they display a less compact demarcation membrane system and a striking inability to form proplatelets. Through megakaryocyte transcriptomics and platelet proteomics, we identified several cytoskeleton-related proteins and downstream effector kinases, including Mylk, that were downregulated upon Sp1/Sp3 depletion, providing an explanation for the observed defects in megakaryopoiesis. Supporting this notion, selective Mylk inhibition by ML7 affected proplatelet formation and stabilization and resulted in defective ITAM receptor-mediated platelet aggregation.
authors.affiliation: Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands;
authors.forename: Marjolein
authors.initials: M
authors.lastname: Meinders
authors.affiliation: Department of Cell Biology, Erasmus MC, Rotterdam, The Netherlands;
authors.forename: Divine I
authors.initials: DI
authors.lastname: Kulu
authors.affiliation: Department of Cell Biology, Erasmus MC, Rotterdam, The Netherlands;
authors.forename: Harmen J G
authors.initials: HJ
authors.lastname: van de Werken
authors.affiliation: Department of Molecular Cell Biology, Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands;
authors.forename: Mark
authors.initials: M
authors.lastname: Hoogenboezem
authors.affiliation: Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands;
authors.forename: Hans
authors.initials: H
authors.lastname: Janssen
authors.affiliation: Center for Biomics, and.
authors.forename: Rutger W W
authors.initials: RW
authors.lastname: Brouwer
authors.affiliation: Center for Biomics, and.
authors.forename: Wilfred F J
authors.initials: WF
authors.lastname: van Ijcken
authors.affiliation: Proteomics Center, Erasmus MC, Rotterdam, The Netherlands; and.
authors.forename: Erik-Jan
authors.initials: EJ
authors.lastname: Rijkers
authors.affiliation: Proteomics Center, Erasmus MC, Rotterdam, The Netherlands; and.
authors.forename: Jeroen A A
authors.initials: JA
authors.lastname: Demmers
authors.affiliation: Institute of Molecular Biology and Tumor Research, Philipps-University Marburg, Marburg, Germany.
authors.forename: Imme
authors.initials: I
authors.lastname: Krüger
authors.affiliation: Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands;
authors.forename: Timo K
authors.initials: TK
authors.lastname: van den Berg
authors.affiliation: Institute of Molecular Biology and Tumor Research, Philipps-University Marburg, Marburg, Germany.
authors.forename: Guntram
authors.initials: G
authors.lastname: Suske
authors.affiliation: Department of Blood Cell Research, Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands;
authors.forename: Laura
authors.identifier: http://orcid.org/0000-0001-8443-900X
authors.initials: L
authors.lastname: Gutiérrez
authors.affiliation: Department of Cell Biology, Erasmus MC, Rotterdam, The Netherlands;
authors.forename: Sjaak
authors.initials: S
authors.lastname: Philipsen
mesh_terms: D000818:Animals; D001381:Azepines / Q000737:chemistry; D001792:Blood Platelets / Q000166:cytology* / Q000378:metabolism; D001853:Bone Marrow / Q000378:metabolism; D005434:Flow Cytometry; D037181:Lectins, C-Type / Q000378:metabolism; D008533:Megakaryocytes / Q000166:cytology*; D051379:Mice; D018345:Mice, Knockout; D009281:Naphthalenes / Q000737:chemistry; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D020543:Proteome; D015398:Signal Transduction; D016329:Sp1 Transcription Factor / Q000235:genetics* / Q000378:metabolism; D051705:Sp3 Transcription Factor / Q000235:genetics* / Q000378:metabolism; D013154:Spleen / Q000378:metabolism; D013921:Thrombocytopenia / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D001381:Azepines; C529518:CLEC-2 protein, mouse; D037181:Lectins, C-Type; D009281:Naphthalenes; D010980:Platelet Membrane Glycoproteins; D020543:Proteome; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; C098341:platelet membrane glycoprotein VI; C070571:ML 7; D051705:Sp3 Transcription Factor