Li Mei, Kellems Rodney E
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas 77030, USA.
Biol Reprod. 2003 Oct;69(4):1220-30. doi: 10.1095/biolreprod.103.015545. Epub 2003 Jun 11.
AP-2gamma is a member of the AP-2 transcription factor family, is highly enriched in the trophoblast cell lineage, and is essential for placental development. In an effort to identify factors regulating AP-2gamma gene expression, we isolated and characterized the promoter and 5'-flanking region of the mouse and human AP-2gamma genes. The transcription start site of the mouse AP-2gamma gene was mapped by primer extension and 5' rapid amplification of cDNA ends. Deletion analysis of the 5'-flanking region revealed a 704-base pair (bp) sequence located approximately 6 kilobases (kb) upstream of the transcription start site that is required for enhanced expression in trophoblast cells. Additional gene transfer studies showed that basal promoter activity resides within a highly conserved, approximately 200-bp DNA sequence located immediately upstream of the transcription start site. The conserved region is highly GC-rich and lacks typical TATA or CCAAT boxes. Multiple potential Sp- and AP-2-binding sites are clustered within this region. Electrophoretic mobility shift assays demonstrated that Sp1 and Sp3 bind to three sites in the promoter region of the mouse AP-2gamma gene. Combined mutation of the three putative Sp sites reduced promoter activity by 80% in trophoblast and nontrophoblast cells, demonstrating the functional importance of these sites in regulating AP-2gamma gene expression. In summary, we have identified a potential trophoblast cell-specific regulatory element located approximately 6 kb upstream of the murine AP-2gamma gene transcription start site, and we have shown that Sp1 and Sp3 bind to cis-regulatory elements located in the promoter proximal region and contribute to basal promoter activity.
AP-2γ是AP-2转录因子家族的成员，在滋养层细胞谱系中高度富集，对胎盘发育至关重要。为了确定调节AP-2γ基因表达的因素，我们分离并鉴定了小鼠和人类AP-2γ基因的启动子及5'侧翼区域。通过引物延伸和cDNA末端的5'快速扩增来定位小鼠AP-2γ基因的转录起始位点。对5'侧翼区域的缺失分析揭示了一个位于转录起始位点上游约6千碱基（kb）处的704碱基对（bp）序列，该序列是滋养层细胞中增强表达所必需的。额外的基因转移研究表明，基础启动子活性位于转录起始位点紧邻上游的一个高度保守的约200 bp DNA序列内。该保守区域富含GC且缺乏典型的TATA或CCAAT框。多个潜在的Sp和AP-2结合位点聚集在该区域内。电泳迁移率变动分析表明，Sp1和Sp3与小鼠AP-2γ基因启动子区域的三个位点结合。这三个假定的Sp位点的联合突变使滋养层细胞和非滋养层细胞中的启动子活性降低了80%，证明了这些位点在调节AP-2γ基因表达中的功能重要性。总之，我们确定了一个位于小鼠AP-2γ基因转录起始位点上游约6 kb处的潜在滋养层细胞特异性调节元件，并且我们已经表明Sp1和Sp3与位于启动子近端区域的顺式调节元件结合并有助于基础启动子活性。
