Dnmt3b ablation impairs fracture repair through upregulation of Notch pathway
Jun Ying, Taotao Xu, Cuicui Wang, Hongting Jin, Peijian Tong, Jianjun Guan, Yousef Abu-Amer, Regis O’Keefe, Jie Shen
Jun Ying, Taotao Xu, Cuicui Wang, Hongting Jin, Peijian Tong, Jianjun Guan, Yousef Abu-Amer, Regis O’Keefe, Jie Shen
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Research Article Bone biology

Dnmt3b ablation impairs fracture repair through upregulation of Notch pathway

Abstract

We previously established that DNA methyltransferase 3b (Dnmt3b) is the sole Dnmt responsive to fracture repair and that Dnmt3b expression is induced in progenitor cells during fracture repair. In the current study, we confirmed that Dnmt3b ablation in mesenchymal progenitor cells (MPCs) resulted in impaired endochondral ossification, delayed fracture repair, and reduced mechanical strength of the newly formed bone in Prx1-Cre;Dnmt3bf/f (Dnmt3bPrx1) mice. Mechanistically, deletion of Dnmt3b in MPCs led to reduced chondrogenic and osteogenic differentiation in vitro. We further identified Rbpjκ as a downstream target of Dnmt3b in MPCs. In fact, we located 2 Dnmt3b binding sites in the murine proximal Rbpjκ promoter and gene body and confirmed Dnmt3b interaction with the 2 binding sites by ChIP assays. Luciferase assays showed functional utilization of the Dnmt3b binding sites in murine C3H10T1/2 cells. Importantly, we showed that the MPC differentiation defect observed in Dnmt3b deficiency cells was due to the upregulation of Rbpjκ, evident by restored MPC differentiation upon Rbpjκ inhibition. Consistent with in vitro findings, Rbpjκ blockage via dual antiplatelet therapy reversed the differentiation defect and accelerated fracture repair in Dnmt3bPrx1 mice. Collectively, our data suggest that Dnmt3b suppresses Notch signaling during MPC differentiation and is necessary for normal fracture repair.

Authors

Jun Ying, Taotao Xu, Cuicui Wang, Hongting Jin, Peijian Tong, Jianjun Guan, Yousef Abu-Amer, Regis O’Keefe, Jie Shen

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Figure 5

Rbpjκ is a target of Dnmt3b.

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Rbpjκ is a target of Dnmt3b. (A) A schematic representing 2 Dnmt3b bindi...
(A) A schematic representing 2 Dnmt3b binding sites (CpG islands, green box) identified in Rbpjκ gene. (B) Pull-down of genomic DNA with a Dnmt3b antibody (ChIP assay) shows interaction of Dnmt3b with its binding site in the Rbpjκ promoter region by qPCR utilizing specific primers (n = 3). (C) Rbpjk promoter luciferase assay was performed in C3H10T1/2 cells with Dnmt3b loss of function (LOF) and gain of function (GOF) (n = 3). (D) Real-time qPCR analysis of Rbpjk, HeyL, Sox9, and Sp7 in C3H10T1/2 cells with Dnm3b LOF and Rbpjκ LOF. The mRNA levels were normalized to that of Actb and then were normalized to the control group (n = 3). (E) Alcian blue and alkaline phosphatase (ALP) stainings were performed to examine the chondrogenic and osteogenic processes, respectively (n = 3). (F) Alcian blue and ALP intensity was measured using NIH ImageJ software (n = 3). (G) Real-time qPCR analyses were performed to determine the relative expression of Agc1, Col2, Alp, and Spp1 in C3H10T1/2 cells. The mRNA levels were normalized to that of Actb and then were normalized to the control group. Data are presented as mean ± SD. *P < 0.05 by 1-way ANOVA test.