Characterization of the complete genomic structure of the human WNT-5A gene, functional analysis of its promoter, chromosomal mapping, and expression in early human embryogenesis
Characterization of the complete genomic structure of the human WNT-5A gene, functional analysis of its promoter, chromosomal mapping, and expression in early human embryogenesis
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DOI：
10.1074/jbc.270.52.31225
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发表时间：
1995-12-29
影响因子：
4.8
通讯作者：
Iozzo, RV
中科院分区：
文献类型：
--
作者：
Danielson, KG;Pillarisetti, J;Iozzo, RV
我们报告了人类WNT-5A基因的完整基因组结构，该基因编码一种富含半胱氨酸的生长因子，参与生长和分化过程中的细胞-细胞信号传导。该基因由5个外显子组成，末端外显子编码约6.5个腺苷酸酶对的大的3 ′-非翻译区，并利用多聚腺苷酸化信号产生至少4个离散的转录物。我们发现了一个新的前导外显子，该外显子被一个411碱基对的内含子中断，该内含子保留在我们原始的cDNA克隆中，启动子区位于富含GpC的岛上，含有大量的顺式作用元件，包括几个GC盒和Sp1，AP 1和AP 2结合基序，它缺乏管家和生长因子基因典型的TATA或CAAT盒，瞬时细胞转染试验显示3，g-双磷酸酶对5 ′-侧翼区具有功能性启动子活性。有趣的是，内部和5 ′-缺失显示远端启动子不是完全转录活性所必需的，并且WNT-5A的前631个碱基对具有最强的启动子活性。使用一组携带染色体3 p部分的啮齿动物-人类杂交DNA，我们将该基因定位于3p14.2-p21.1，位于体质性和家族性肾细胞癌相关易位之间，对妊娠28-42天的早期人类胚胎的原位杂交分析显示，WNT-5A转录本不限于发育中的大脑和四肢，但也观察到在间充质接壤的咽裂和囊，并在发育中的性腺和肾脏。相对较高的表达在celomic上皮细胞和卵泡和曲细精管的前体表明一个新的作用WNT-5A在生殖细胞分化。这项研究为识别WNT-5A基因的调控提供了分子基础，并为研究与这一重要基因相关的遗传疾病提供了机会。
We report the complete genomic organization of the human WNT-5A gene, which encodes a cysteine-rich growth factor involved in cell-cell signaling during growth and differentiation. The gene comprises five exons with the terminal exon coding for a large 3'-untranslated region of approximate to 6.5 kilobase pairs and utilizes multiple polyadenylation signals to generate at least four discrete transcripts, We discovered a new leader exon interrupted by a 411-base pair intron that was retained in our original cDNA cloning, The promoter region was located in a GpC-rich island and harbored numerous cis-acting elements including several GC boxes and Sp1, AP1, and AP2 binding motifs, It lacked TATA or CAAT boxes typical of housekeeping and growth factor genes, In support of this, primer extension revealed two transcription start sites, Transient cell transfection assays showed functional promoter activity for the 3,g-kilobase pair 5'-flanking region, Interestingly, internal and 5' deletions revealed that the distal promoter was not required for full transcriptional activity and that the first 631 base pairs of WNT-5A harbored the strongest promoter activity. Using a panel of rodent-human hybrid DNAs carrying portions of chromosome 3p, we mapped the gene to 3p14.2-p21.1, between a constitutional and a familial renal cell carcinoma-associated translocation, In situ hybridization analyses of early human embryos at 28-42 days of gestation revealed that WNT-5A transcripts were not restricted to the developing brain and limbs but were also observed in the mesenchyme bordering the pharyngeal clefts and pouches and in the developing gonads and kidneys. The relatively high expression in the celomic epithelium and in the precursors of follicles and seminiferous tubules suggest a novel role for WNT-5A in germ-cell differentiation. This study provides the molecular basis for discerning the regulation of the WNT-5A gene and offers the opportunity to investigate genetic disorders linked to this important gene.