G9a is involved in the regulation of cranial bone formation through activation of Runx2 function during development
G9a is involved in the regulation of cranial bone formation through activation of Runx2 function during development
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DOI：
10.1016/j.bone.2020.115332
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发表时间：
2020-08-01
期刊：
影响因子：
4.1
通讯作者：
Nifuji, Akira
中科院分区：
文献类型：
--
作者：
Ideno, Hisashi;Nakashima, Kazuhisa;Nifuji, Akira
甲基转移酶G9a最初是作为一种组蛋白甲基转移酶分离出来的，它可以催化组蛋白3赖氨酸9 （H3K9）甲基化到二甲基化状态（H3K9me2）。最近的研究表明，G9a在包括成骨细胞在内的多种细胞中具有多种功能。在这里，我们研究了G9a在颅骨形成过程中的功能。将Sox9-cre与G9a(flox/flox) （(fl/fl)）小鼠杂交，产生在sox9阳性神经嵴源性骨细胞中缺乏G9a表达的条件敲除小鼠。Sox9-Cre/G9a（fl/fl）小鼠颅骨拱顶骨出现严重的低矿化，包括鼻骨、额骨和顶骨缺损，并伴有囟门打开。g9a缺失后，颅骨骨组织细胞增殖受到抑制。骨标记基因，即碱性磷酸酶和骨钙素的表达水平受到抑制，而Runx2的表达在这些组织中没有明显降低。体外实验显示，G9a缺失后的颅骨成骨细胞增殖能力下降。在g9a缺失的成骨细胞中，成纤维细胞生长因子受体和几个细胞周期蛋白的表达水平受到抑制。此外，骨标记基因的表达减少，而Runx2的表达未因G9a缺失而改变。G9a增强了Runx2的转录活性，而靶向G9a的siRNA抑制了Runx2在C3H10T1/2间充质细胞中的转录活性。我们证实了内源性Runx2与G9a的直接关联。染色质免疫沉淀实验表明，G9a与初代成骨细胞启动子runx2靶区结合。此外，在g9缺失的成骨细胞中，Runx2与骨钙素启动子的结合被破坏。上述结果提示G9a通过与Runx2结合并激活Runx2调控颅骨骨细胞的增殖和分化。
The methyltransferase G9a was originally isolated as a histone methyltransferase that catalyzes the methylation of histone 3 lysine 9 (H3K9) to a dimethylated state (H3K9me2). Recent studies have revealed that G9a has multiple functions in various cells, including osteoblasts. Here, we investigated G9a function during cranial bone formation. Crossing Sox9-cre with G9a(flox/flox) ((fl/fl)) mice generated conditional knockout mice lacking G9a expression in Sox9-positive neural crest-derived bone cells. Sox9-Cre/G9a(fl/fl) mice showed severe hypo-mineralization of cranial vault bones, including defects in nasal, frontal, and parietal bones with opened fontanelles. Cell proliferation was inhibited in G9a-deleted calvarial bone tissues. Expression levels of bone marker genes, i.e., alkaline phosphatase and osteocalcin, were suppressed, whereas Runx2 expression was not significantly decreased in those tissues. In vitro experiments using G9a-deleted calvarial osteoblasts showed decreased cell proliferation after G9a deletion. In G9a-deleted osteoblasts, expression levels of fibroblast growth factor receptors and several cyclins were suppressed. Moreover, the expression of bone marker genes was decreased, whereas Runx2 expression was not altered by G9a deletion in vitro. G9a enhanced the transcriptional activity of Runx2, whereas siRNA targeting G9a inhibited the transcriptional activity of Runx2 in C3H10T1/2 mesenchymal cells. We confirmed the direct association of endogenous Runx2 with G9a. Chromatin immunoprecipitation experiments showed that G9a bound to Runx2-target regions in promoters in primary osteoblasts. Furthermore, Runx2 binding to the osteocalcin promoter was abrogated in G9-deleted osteoblasts. These results suggest that G9a regulates proliferation and differentiation of cranial bone cells through binding to and activating Runx2.