Unezaki Sawako, Nishizawa Mikio, Okuda-Ashitaka Emiko, Masu Yasuo, Mukai Masanori, Kobayashi Satoru, Sawamoto Kazunobu, Okano Hideyuki, Ito Seiji
Department of Medical Chemistry, Kansai Medical University, 10-15 Fumizono, Moriguchi, Osaka 570-8506 Japan.
Gene. 2004 Jul 7;336(1):47-58. doi: 10.1016/j.gene.2004.03.013.
We previously described two isoforms (MOVO-A and -B) of a novel zinc finger protein MOVO, a mouse homologue of Drosophila Ovo protein. Here, we isolated cDNA encoding the third isoform MOVO-C, which had a transactivation domain and zinc finger domain, but lacked an N-terminal potential repression domain that was present in MOVO-A. Three isoform mRNAs were expressed highly in mouse testis and also in the ovary at lower levels. The structural analyses of the isolated Movo gene and mRNAs demonstrated that three different Movo transcripts were differentially processed to generate three isoforms. Major mRNA species encoded MOVO-B with a zinc finger domain alone, and minor mRNA species encoded MOVO-A (potential repressor) and MOVO-C (potential activator). To assign MOVO to a transcriptional factor, we characterized DNA-binding and transactivation properties. Random oligonucleotide selection, electrophoretic mobility shift assay and footprinting indicated that MOVO bound to the sequence, 5'-G(G/C/T)GGGGG-3'. These motifs were found in the 5'-flanking regions of Movo and other testis-specific genes. Nuclear proteins binding to this motif were detected in mouse testis, and the expression of MOVO mRNA was restricted in spermatocytes. The luciferase assay demonstrated that MOVO-C activated Movo promoter and MOVO-A repressed it, but MOVO-B had no effects. Mutated MOVO-binding motifs in the Movo promoter reduced the luciferase activity. All the isoforms had no effects on SV40 promoter without MOVO-binding motifs. MOVO-A partially rescued oogenesis of a Drosophila ovo mutant. These results suggest that MOVO isoforms are transcription factors to regulate genes carrying the MOVO-binding motifs in the testis.
我们之前描述了一种新型锌指蛋白MOVO的两种异构体（MOVO-A和-B），它是果蝇Ovo蛋白的小鼠同源物。在此，我们分离出了编码第三种异构体MOVO-C的cDNA，其具有反式激活结构域和锌指结构域，但缺少MOVO-A中存在的N端潜在抑制结构域。三种异构体mRNA在小鼠睾丸中高表达，在卵巢中也有较低水平的表达。对分离出的Movo基因和mRNA的结构分析表明，三种不同的Movo转录本经过不同的加工产生了三种异构体。主要的mRNA种类仅编码具有锌指结构域的MOVO-B，次要的mRNA种类编码MOVO-A（潜在阻遏物）和MOVO-C（潜在激活物）。为了将MOVO归类为转录因子，我们对其DNA结合和反式激活特性进行了表征。随机寡核苷酸选择、电泳迁移率变动分析和足迹法表明，MOVO与序列5'-G(G/C/T)GGGGG-3'结合。这些基序存在于Movo和其他睾丸特异性基因的5'侧翼区域。在小鼠睾丸中检测到与该基序结合的核蛋白，并且MOVO mRNA的表达在精母细胞中受到限制。荧光素酶测定表明，MOVO-C激活Movo启动子，而MOVO-A抑制它，但MOVO-B没有作用。Movo启动子中突变的MOVO结合基序降低了荧光素酶活性。所有异构体对没有MOVO结合基序的SV40启动子均无作用。MOVO-A部分挽救了果蝇ovo突变体的卵子发生。这些结果表明，MOVO异构体是调节睾丸中携带MOVO结合基序基因的转录因子。
