Mastermind-like domain-containing 1 (MAMLD1 or CXorf6) transactivates the Hes3 promoter, augments testosterone production, and contains the SF1 target sequence
Mastermind-like domain-containing 1 (MAMLD1 or CXorf6) transactivates the Hes3 promoter, augments testosterone production, and contains the SF1 target sequence
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DOI：
10.1074/jbc.m703289200
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发表时间：
2008-02-29
影响因子：
4.8
通讯作者：
Ogata, Tsutomu
中科院分区：
文献类型：
--
作者：
Fukami, Maki;Wada, Yuka;Ogata, Tsutomu
虽然X染色体开放阅读框6 （xorf6）已被证明是尿道下裂的致病基因，但其分子功能尚不清楚。为了澄清这一点，我们首先检查了CXorf6蛋白结构，确定了其与mastermind-like 2 （MAML2）蛋白的同源性，MAML2蛋白在典型Notch信号传导中起协同激活作用。荧光素酶对野生型CXorf6蛋白的反激活分析表明，CXorf6显著反激活了非典型Notch靶基因Hes3 （hairy/enhancer of split 3）的启动子，但没有明显的dna结合能力。对上述三个明显病理性的无义突变进行了转激活分析，表明E124X和Q197X蛋白没有转激活功能，而R653X蛋白保留了近乎正常的转激活功能。亚细胞定位分析显示，野生型和R653X蛋白与MAML2蛋白在核体中共定位，而E124X和Q197X蛋白不能定位到核体。因此，我们对R653X进行了进一步的研究，揭示了无义介导的mRNA衰变在体内的发生。接下来，使用小干扰RNA进行短暂敲低xorf6，显示小鼠间质瘤细胞中睾酮产生减少。此外，甾体生成因子1（甾体生成因子1，steriidogenic factor 1, SF1）蛋白结合到CXorf6编码区上游的特定序列上，并发挥了交互激活活性。这些结果表明，CXorf6可激活Hes3启动子，增加睾酮的产生，并含有SF1靶序列，从而为阐明CXorf6的生物学作用提供了第一个线索。基于其特征结构，我们将xorf6命名为MAMLD1 （mastermind-like domain-containing 1）。
Although chromosome X open reading frame 6 (CXorf6) has been shown to be a causative gene for hypospadias, its molecular function remains unknown. To clarify this, we first examined CXorf6 protein structure, identifying homology to mastermind-like 2 (MAML2) protein, which functions as a co-activator in canonical Notch signaling. Transactivation analysis for wildtype CXorf6 protein by luciferase assays showed that CXorf6 significantly transactivated the promoter of a noncanonical Notch target gene hairy/enhancer of split 3 (Hes3) without demonstrable DNA-binding capacity. Transactivation analysis was also performed for the previously described three apparently pathologic nonsense mutations, indicating that E124X and Q197X proteins had no transactivation function, whereas R653X protein retained a nearly normal transactivation function. Subcellular localization analysis revealed that wild-type and R653X proteins co-localized with MAML2 protein in nuclear bodies, whereas E124X and Q197X proteins were incapable of localizing to nuclear bodies. Thus, further studies were performed for R653X, revealing the occurrence of nonsense mediated mRNA decay in vivo. Next, transient knockdown of CXorf6 was performed using small interfering RNA, showing reduced testosterone production in mouse Leydig tumor cells. Furthermore, steroidogenic factor 1 (SF1) protein bound to a specific sequence in the upstream of the CXorf6 coding region and exerted a transactivation activity. These results suggest that CXorf6 transactivates the Hes3 promoter, augments testosterone production, and contains the SF1 target sequence, thereby providing the first clue to clarify the biological role of CXorf6. We designate CXorf6 as MAMLD1 (mastermind-like domain-containing 1) based on its characteristic structure.