Abstract：
Mef2c is a transcription factor that mediates key cellular behaviors that promote endochondral ossification and bone formation. Previously, Mef2c has been shown to regulate Sost transcription via its osteocyte-specific enhancer, ECR5, and conditional deletions of Mef2cfl/fl with either Col1-Cre or Dmp1-Cre produced generalized high bone mass (HBM) consistent with Van Buchem Disease phenotypes. However, Sost-/-; Mef2cfl/fl; Dmp1-Cre mice produced a significantly higher bone mass phenotype that Sost-/- alone suggesting that Mef2c modulates bone mass through additional mechanisms, independent of Sost. To identify new Mef2c transcriptional targets important in bone metabolism, we profiled gene expression by single-cell RNA sequencing in subpopulations of cells isolated from Mef2cfl/fl; Dmp1-Cre and Mef2cfl/fl; Bglap-Cre femurs, both strains exhibiting similar high bone mass phenotypes. However, we found Mef2cfl/fl; Bglap-Cre to also display a growth plate defect characterized by an expansion of several osteoprogenitor subpopulations. Differential gene expression analysis identified a total of 96 up- and 2434 down- regulated genes in Mef2cfl/fl; Bglap-Cre and 176 up- and 1041 down- regulated genes in Mef2cfl/fl; Dmp1-Cre bone cell subpopulations compared to wildtype mice. Mef2c deletion affected the transcriptomes across several cell types including mesenchymal progenitors (MP), osteoprogenitors (OSP), osteoblast (OB), and osteocyte (OCY) subpopulations. Several energy metabolism genes such as Uqcrb, Ndufv2, Ndufs3, Ndufa13, Ndufb9, Ndufb5, Cox6a1, Cox5a, Atp5o, Atp5g2, Atp5b, Atp5 were significantly down regulated in Mef2c-deficient OBs and OCYs, in both strains. Binding motif analysis of promoter regions of differentially expressed genes identified Mef2c binding in Bone Sialoprotein (BSP/Ibsp), a gene known to cause increased trabecular BV/TV in the femurs of Ibsp-/- mice. Immunohistochemical analysis confirmed the absence of Ibsp protein in OBs and OCYs. These findings suggests that the HBM in Sost-/-; Mef2cfl/fl; Dmp1-Cre is caused by a multitude of transcriptional changes in genes that regulate bone formation, two of which are Sost and Ibsp.
摘要：
Mef2c是介导促进软骨内骨化和骨形成的关键细胞行为的转录因子。以前,Mef2c已被证明通过其骨细胞特异性增强子调节Sost转录，ECR5和Col1-Cre或Dmp1-Cre的Mef2cfl/f的条件性缺失产生了与VanBuchem病表型一致的全身性高骨量（HBM）。然而,Sost-/-;Mef2cfl/fl;Dmp1-Cre小鼠产生了显着更高的骨量表型，Sost-/-单独表明Mef2c通过其他机制调节骨量，独立于Sost。为了鉴定在骨代谢中重要的新的Mef2c转录靶标，我们通过单细胞RNA测序分析了从Mef2cfl/fl分离的细胞亚群中的基因表达；Dmp1-Cre和Mef2cfl/fl；Bglap-Cre股骨，两种菌株表现出相似的高骨量表型。然而,我们发现Mef2cfl/fl;Bglap-Cre也显示出生长板缺陷，其特征是几个骨祖细胞亚群的扩张。差异基因表达分析鉴定了Mef2cfl/fl中总共96个上调和2434个下调基因;Mef2cfl/fl中的Bglap-Cre和176个下调基因;Dmpl-Cre骨细胞亚群与野生型小鼠相比。Mef2c缺失影响了几种细胞类型的转录组，包括间充质祖细胞（MP），骨祖细胞（OSP），成骨细胞(OB),和骨细胞（OCY）亚群。几种能量代谢基因，如Uqcrb，Ndufv2,Ndufs3,Ndufa13,Ndufb9,Ndufb5,Cox6a1,Cox5a,Atp5o,Atp5g2,Atp5b,Atp5在Mef2c缺陷型OBs和OCYs中显著下调，在这两种菌株中。差异表达基因启动子区的结合基序分析确定了骨唾液酸蛋白（BSP/Ibsp）中的Mef2c结合，一种已知会导致Ibsp-/-小鼠股骨小梁BV/TV增加的基因。免疫组织化学分析证实OBs和OCYs中不存在Ibsp蛋白。这些发现表明，Sost-/-;Mef2cfl/fl;Dmp1-Cre中的HBM是由调节骨形成的基因中的多种转录变化引起的，其中两个是Sost和Ibsp。
