Functional Analysis of Placenta-Specific Transcription Factor during Placental Development

我们从人胎盘中分离出一个2.8 kb的cDNA克隆，编码一个436个氨基酸的蛋白，该蛋白决定了人芳香化酶的组织特异性表达！以滋养层细胞特异性元件2为报告序列的酵母单杂交系统cDNA文库。该蛋白被发现与hGCMa相同，hGCMa是果蝇GCM（胶质细胞缺失）的哺乳动物同源物。果蝇GCM是调节神经胶质细胞命运的神经转录因子。hGCMa仅在人胎盘中表达。在Sf - 21细胞中表达的蛋白也与瘦素启动子中的PLE1结合，其他的顺式元件报道了胎盘特异性表达，这表明hGCMa是一种胎盘特异性转录调节剂，可能参与了多个胎盘特异性基因的表达（Yamada K., Ogawa H., et al. 1999 J. Biol）。化学。274:32279-32286)。我们还确定了hGCMa基因的基因组组织、染色体定位和完整的22 kb DNA序列。它包含6个外显子和5个内含子，编码2.8 kb的cDNA。人类和小鼠的整体基因组组织是相似的。在人类基因的5'侧区发现了转录因子如GATA、Oct-1和βHLH蛋白的几个潜在结合位点。GCM蛋白结合的DNA基序存在于5'侧区，与小鼠基因高度同源。利用荧光原位杂交技术将该基因定位到6号染色体上（Yamada K., Ogawa H., et al. 2000）。Biophys。《共同编》278:134-139)。制备兔抗hgcma抗体。在初步实验中，除了胎盘，它还与几种人类血细胞发生反应。现在我们准备制备更特异的抗hGCMa抗体。

We isolated a 2.8 kb cDNA clone encoding a 436-amino acid protein that dictates tissue-specific expression of human aromatase from a human placenta! cDNA library using a yeast one-hybrid system with a trophoblast-specific element 2 as a reporter sequence. The protein was revealed to be identical to hGCMa, a mammalian homologue of the Drosophila GCM (glia cells missing). Drosophila GCM is a neural transcription factor that regulates glial cell fate.Expression of hGCMa is restricted to the placenta in human. The protein expressed in Sf 21 cells also binds to PLE1 in the leptin promoter among other cis-elements reported to confer placenta-specific expression, suggesting that hGCMa is a placenta-specific transcription regulator, possibly involved in the expression of multiple placenta-specific genes (Yamada K., Ogawa H., et al. 1999 J. Biol. Chem. 274: 32279-32286).We also determined the genomic organization, chromosomal localization, and the complete 22 kb DNA sequence of hGCMa gene. It contains six exons and five introns, encoding a 2.8 kb cDNA. Overall genomic organization is similar for the human and mouse. Several potential binding sites for transcription factors like GATA, Oct-1, and βHLH proteins were found in the 5'-flanking region of the human gene. A DNA motif for GCM protein binding exists in the 5'-flanking region that is highly homologous with that of the mouse gene. The location of this gene was mapped to chromosome 6 using fluorescence in situ hybridization (Yamada K., Ogawa H., et al. 2000 Biochem. Biophys. Res. Commun. 278: 134-139).We prepared anti-hGCMa antibody in rabbits. In preliminary experiments, it reacted with also several human blood cells besides the placenta. Now we are going to prepare more specific antibody against hGCMa.