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

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 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. Overall design: 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（Krüppel-like transcription factor）家族。二者同源性极高，广泛表达于各类组织，且可识别富含G碱基的DNA基序（DNA motif）。既往研究认为它们可调控细胞周期与生长调控、代谢通路以及细胞凋亡（apoptosis）等通用生物学过程。在小鼠体内敲除Sp1或Sp3会引发致死表型，而复合单倍体不足（haploinsufficiency）则会导致胎儿阶段出现造血缺陷。本研究证实，在成年小鼠中条件性敲除单个Sp1或Sp3对造血功能仅产生极微弱影响；而同时缺失Sp1与Sp3则会引发严重的巨血小板减少症（macrothrombocytopenia）与血小板功能障碍。我们采用流式细胞术（flow cytometry）、细胞培养与电子显微镜（electron microscopy）技术开展实验，结果显示：尽管骨髓与脾脏中的巨核细胞（megakaryocyte）数量并无异常，但此类细胞的分界膜系统（demarcation membrane system）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（transcriptomics）与血小板蛋白质组学（proteomics）分析，我们鉴定出多个与细胞骨架相关的蛋白以及下游效应激酶（包括Mylk在内），这些分子在Sp1/Sp3缺失后表达下调，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。我们证实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（immunoreceptor tyrosine-based activation motif, ITAM）受体介导的血小板聚集均不可或缺。本研究数据凸显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所兼具的特异性与通用性功能。总体实验设计：通过深度测序（deep sequencing）生成Sp1fl/fl::Sp3fl/fl（WTlox）与Pf4-Cre::Sp1fl/fl::Sp3fl/fl（dKO）小鼠的巨核细胞mRNA表达谱，每组设置三次生物学重复。