Identification of Ube2b as a Novel Target of Androgen Receptor in Mouse Sertoli Cells
Identification of Ube2b as a Novel Target of Androgen Receptor in Mouse Sertoli Cells
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鉴定 Ube2b 作为小鼠支持细胞雄激素受体的新靶标
DOI：
10.1095/biolreprod.112.103648
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发表时间：
2013-08-01
影响因子：
3.6
通讯作者：
Gui, Yaoting
中科院分区：
文献类型：
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作者：
Mou, Lisha;Zhang, Qiaoxia;Gui, Yaoting
许多基因受雄激素及其受体(AR)的调控，但AR的直接靶基因，特别是与精子发生和男性不育有关的基因尚不清楚。在这里，我们确定泛素结合酶e2b(Ube2b)是AR的关键靶基因。定量RT-PCR法和免疫荧光法分析支持细胞AR基因敲除(S-AR(-/y))小鼠睾丸中UBE2B的表达。用Western印迹和qRT-PCR进一步证实了睾酮对小鼠睾丸支持细胞系TM4细胞Ube2b基因的上调作用。此外，荧光素酶分析、电泳迁移率改变分析和染色质免疫沉淀分析证实，配体结合的AR通过直接与Ube2b启动子的雄激素反应元件结合而激活Ube2b转录。体外分析表明，睾酮增加了UBE2B的表达并激活了H_2A泛素化，而下调UBE2B则阻断了睾酮诱导的H_2A泛素化。免疫组织化学显示S-AR(-/y)小鼠睾丸组织中H_2A泛素化水平明显降低。数字基因表达分析显示，UBE2B显著下调TM4细胞113个基因，上调71个基因。这些结果表明，Ube2b作为Sertoli细胞中AR的直接转录靶点，通过促进H_2A泛素化来介导AR在精子发生中的作用。
Many genes are regulated by androgen and its receptor (AR), but the direct target genes of AR, especially those involved in spermatogenesis and male infertility, remain unclear. Here, we identified ubiquitin-conjugating enzyme E2B (Ube2b) as a critical target gene of AR. The expression of UBE2B was decreased in the testes of Sertoli cell AR knockout (S-AR(-/y)) mice analyzed by quantitative RT-PCR (qRT-PCR) and immunofluorescence. The upregulation of Ube2b gene by testosterone was further demonstrated by Western blot and qRT-PCR in TM4 cells, a mouse Sertoli cell line. Moreover, luciferase assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay validated that the ligand-bound AR activated Ube2b transcription via direct binding to the androgen-responsive element of the Ube2b promoter. In vitro analyses showed that testosterone increased UBE2B expression and activated H2A ubiquitylation, while downregulation of UBE2B blocked the testosterone-induced H2A ubiquitylation. The ubiquitylation of H2A was markedly decreased in the testes of S-AR(-/y) mice by immunohistochemistry. Digital gene expression analysis showed that 113 genes were significantly downregulated and 71 were upregulated by UBE2B in TM4 cells. These results suggest that Ube2b, as a direct AR transcriptional target in Sertoli cells, mediates the function of AR in spermatogenesis by promoting H2A ubiquitylation.