CCAAT/enhancer binding protein beta regulates expression of the cystatin-related epididymal spermatogenic (Cres) gene.
CCAAT/enhancer binding protein beta regulates expression of the cystatin-related epididymal spermatogenic (Cres) gene.
复制标题
CCAAT/增强子结合蛋白β调节半胱氨酸蛋白酶抑制剂相关的附睾生精（Cres）基因的表达。
DOI：
10.1095/biolreprod65.5.1452
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发表时间：
2001
影响因子：
3.6
通讯作者：
Cornwall,GA
中科院分区：
文献类型：
--
作者：
Hsia,N;Cornwall,GA
克雷斯蛋白是半胱氨酸蛋白酶抑制剂的胱抑素超家族的成员，在阶段特异性生殖细胞、附睾近端头和垂体前叶促性腺激素细胞中具有限制性表达。为了阐明调节该基因高度限制性表达的分子机制，我们对小鼠res 5 ′侧翼序列的1.6个碱基进行了测序，并对resgene启动子进行了研究。在cres启动子的前135个碱基对内存在两个推测的CCAAT/增强子结合蛋白（C/EBP）转录因子结合基序。此外，我们的研究表明，在C/EBPβ缺陷小鼠的附睾中，cresmRNA水平显著降低。这些数据表明，C/EBP家族的转录因子，特别是C/EBPβ，在调节cresgene表达中起作用。北方印迹分析表明，C/EBPβ是LβT2促性腺细胞系和附睾近端头中表达的主要C/EBP家族成员。凝胶迁移和超迁移实验表明，LβT2细胞和附睾细胞核提取物中的C/EBPβ蛋白与启动子上的两个C/EBP位点结合。最后，为了检测C/EBP位点增加基因表达的体内功能，在LβT2促性腺激素细胞和两种异源细胞系统中进行了转染研究。这些实验表明，当C/EBP位点突变时，cres启动子的transC/EBP激活显著降低，而当两个C/EBP位点都突变时，cres启动子没有transC/EBP激活。综上所述，这些研究表明C/EBPβ转录因子是近端附睾头和垂体前叶促性腺激素细胞高水平表达cresgene所必需的。
The CRES protein is a member of the cystatin superfamily of cysteine protease inhibitors with restricted expression in stage-specific germ cells, proximal caput epididymidis, and anterior pituitary gonadotroph cells. To elucidate the molecular mechanisms regulating the highly restricted expression of thecresgene, we have sequenced 1.6 kilobases of mousecres5′ flanking sequence and performed studies to examine thecresgene promoter. Two putative CCAAT/enhancer binding protein (C/EBP) transcription factor binding motifs exist within the first 135 base pairs ofcrespromoter. Furthermore, our studies demonstrate thatcresmRNA levels are dramatically reduced in the epididymides of C/EBPβ-deficient mice. These data suggest that the C/EBP family of transcription factors, in particular C/EBPβ, plays a role in the regulation ofcresgene expression. In support of this finding, Northern blot analysis showed that C/EBPβ is the predominant C/EBP family member expressed in the LβT2 gonadotroph cell line and the proximal caput epididymidis. Also, gel shift and supershift assays demonstrated that C/EBPβ protein in nuclear extracts from LβT2 gonadotroph cells and epididymal cells bound to the two C/EBP sites in thecrespromoter. Finally, to test the in vivo function of the C/EBP sites incresgene expression, transfection studies were performed in LβT2 gonadotroph cells and two heterologous cell systems. These experiments showed a significant reduction ofcrestransactivation when either C/EBP sites were mutated, and no transC/EBP activation of thecrespromoter when both C/EBP sites were mutated. Taken together, these studies demonstrate that the C/EBPβ transcription factor is necessary for high levels ofcresgene expression in the proximal caput epididymidis and anterior pituitary gonadotroph cells.