TRANSCIPTIONAL MECHANISM OF DF3 ANTIGEN GENE EXPRESSION

DF3抗原基因表达的转录机制

• 批准号: 2099087
• 负责人: MIYAKO ABE
• 金额: $ 11.46万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2099087
• 关键词: DNA binding protein; DNA footprinting; beta galactosidase; breast neoplasm /cancer diagnosis; breast neoplasms; calorimetry; cell differentiation; differentiation antigens; gene deletion mutation; gene expression; genetic enhancer element; genetic library; genetic promoter element; genetic regulatory element; genetic transcription; glycoproteins; immunoperoxidase; mammary epithelium; molecular cloning; monoclonal antibody; nucleic acid repetitive sequence; polymerase chain reaction; protein sequence; radiotracer; transcription factor; transfection

This proposal addresses mechanisms responsible for regulating expression of the DF3 glycoprotein, a differentiation antigen which is aberrantly expressed in malignant breast epithelial cells. Monoclonal antibody (MAb) DF3 detects a family of mucin-like glycoproteins with high molecular weights (300-500 kD) which are expressed in a polymorphic fashion. Immunoperoxidase staining with MAb DF3 distinguishes malignant and benign breast lesions. DF3 antigen is detectable on the apical borders of secretory mammary epithelial cells and is over-expressed in the cytosol in less differentiated malignant cells. Circulating DF3 antigen levels can be used to monitor the clinical course of breast cancer patients. The level of DF3 antigen expression has also been shown to correlate with degree of breast tumor differentiation. The cDNA encoding the DF3 protein has been cloned as well as 1.6 kb upstream to the transcription start site. A large proportion of the cDNA is occupied by GC-rich 60 bp tandem repeats. These repeats code for 20 amino acid tandems rich in serine, threonine, proline, alanine and glycine. Polymorphic expression of DF3 antigen results from genes containing variable number of tandem repeats. Previous studies have demonstrated that DF3 gene expression is regulated at the transcriptional level. I have recently identified an element located at position -505 to -485 in the DF3 promoter that is functional in regulating DF3 gene transcription. this element functions in both orientations in DF3 promoter and enhancer constructs. This sequence 5'- (GGGAAGTGGTGGGGGGAGGGA) has not been previously described as a cis- element. Moreover, I have found that this sequence specifically interacts with a 45 kD nuclear protein. The objectives of this proposal are to further define the regulatory mechanisms of DF3 gene transcription, characterize the regulatory proteins and study the involvement of these proteins in the aberrant expression of DF3 antigen. The specific aims are: 1) to further define the sequences which bind to nuclear protein(s) that regulate DF3 transcription; 2) to determine the nuclear protein(s) which bind to functional cis-elements that regulate DF3 transcription; 3) to clone the gene(s) coding for DF3 regulatory nuclear protein(s); 4) to determine whether levels of DF3 regulatory protein(s) correspond with aberrant expression of DF3 antigen in human mammary epithelial cells.

这项建议涉及负责管制言论的机制 DF 3糖蛋白是一种分化抗原， 在恶性乳腺上皮细胞中表达。 单克隆抗体（MAb）DF 3检测粘蛋白样蛋白家族 具有高分子量（300-500 kD）的糖蛋白， 以多态的方式表达。 单克隆抗体免疫过氧化物酶染色 DF 3区分恶性和良性乳腺病变。 DF 3抗原是 在分泌性乳腺上皮细胞的顶端边缘可检测到 并且在低分化恶性肿瘤的细胞质中过度表达。 细胞 循环DF 3抗原水平可用于监测 乳腺癌患者的临床过程。 DF 3抗原水平 表达也显示与乳腺肿瘤的程度相关， 分化 已克隆了编码DF 3蛋白的cDNA，其大小为1.6kb， 转录起始位点的上游。 大部分的cDNA 由富含GC的60 bp串联重复序列占据。 这些重复代码为20 富含丝氨酸、苏氨酸、脯氨酸、丙氨酸和 甘氨酸 DF 3抗原的基因多态性表达 含有可变数目的串联重复序列。 以往的研究表明，DF 3基因的表达受到调控， 在转录水平上。 我最近发现了一种元素 位于DF 3启动子中的-505至-485位， 调节DF 3基因转录。 该元件在两种情况下都起作用 在DF 3启动子和增强子构建体中的定向。 这个序列5 '- （GGGAAGTGGTGGGGGGAGGGA）先前未被描述为顺式- 元素 此外，我发现这个序列 与45 kD核蛋白相互作用。 该提案的目的是进一步明确监管 DF 3基因转录机制，表征调控 蛋白质，并研究这些蛋白质参与异常的 DF 3抗原的表达。 具体目标是：1）进一步明确 与调节DF 3的核蛋白结合的序列 转录; 2）确定结合至 调节DF 3转录的功能性顺式元件; 3）克隆 编码DF 3调节核蛋白的基因; 4）确定 DF 3调节蛋白的水平是否对应于异常的 DF 3抗原在人乳腺上皮细胞中的表达。