Sp1/Sp3 transcription factors regulate hallmarks of megakaryocyte maturation, and platelet formation and function

Sp1 and Sp3 belong to the Specificity proteins (Sp)/Kruppel-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 ablation of either Sp1 or Sp3 has minimal impact on hematopoiesis, while the simultaneous loss of Sp1 and Sp3 results in severe macrothrombocytopenia and platelet dysfunction. 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. We show that Mylk is required for proplatelet formation and stabilization and for ITAM-receptor mediated platelet aggregation. Our data highlights the specific vs generic role of these ubiquitous transcription factors in the highly specialized megakaryocytic lineage. Megakaryocyte mRNA profiles of Sp1fl/fl::Sp3fl/fl (WTlox) and Pf4-Cre::Sp1fl/fl::Sp3fl/fl (dKO) mice were generated by deep sequencing, in triplicate.Sp1与Sp3隶属于特异性蛋白（Specificity proteins, Sp）/ Krüppel样转录因子家族，二者亲缘关系紧密，呈广泛表达模式，可识别富含G的DNA基序。现有研究认为，它们可调控细胞周期与生长调控、代谢通路及细胞凋亡等通用生物学过程。在小鼠中敲除Sp1或Sp3会引发致死表型，而复合单倍剂量不足则会导致胎儿阶段的造血缺陷。本研究显示，在成年小鼠中单独条件性敲除Sp1或Sp3对造血功能影响极小，但同时缺失Sp1与Sp3则会引发严重的巨血小板减少症与血小板功能异常。本研究通过流式细胞术、细胞培养及电子显微镜实验发现，尽管骨髓与脾脏中的巨核细胞（megakaryocyte）数量正常，但其分界膜系统结构更为松散，且显著无法形成前血小板（proplatelet）。通过巨核细胞转录组学与血小板蛋白质组学分析，本研究鉴定出多个与细胞骨架相关的蛋白及下游效应激酶，包括肌球蛋白轻链激酶（Mylk），在Sp1/Sp3缺失后这些分子的表达均出现下调，这为巨核细胞生成（megakaryopoiesis）过程中观察到的缺陷提供了合理解释。研究证实，Mylk对于前血小板的形成与稳定，以及免疫受体酪氨酸活化基序（ITAM）受体介导的血小板聚集均不可或缺。本研究数据揭示了这类广泛表达的转录因子在高度特化的巨核细胞谱系中，分别发挥特异性与通用性功能的差异。 本研究对Sp1fl/fl::Sp3fl/fl（WTlox）与Pf4-Cre::Sp1fl/fl::Sp3fl/fl（dKO）小鼠的巨核细胞mRNA转录谱进行了深度测序，每组设置3次生物学重复。