ANALYSIS OF THE GENE PROMOTERS OF HUMAN BONE MORPHOGENETIC PROTEINS

骨形态发生蛋白(BMPs)最早被认为是诱导异质骨和软骨组织的因子，现在被认为在胚胎发育的器官发生中发挥着重要作用。BMPs的氨基酸序列从果蝇到哺乳动物都被认为是非常保守的。到目前为止，BMP根据其氨基酸全序列被分为几个亚类。然而，对于每个BMP的生理作用和功能共享知之甚少。在本研究中，我们试图通过分析每个BMP基因的启动子区域来表征每个BMP的功能共享，而不考虑最近基于氨基酸同源性的分类系统。5‘-侧翼区是一种启动子结构，目前主要用于研究特定靶基因的基因表达调控机制。通过对启动子区域的包含性分子克隆，我们扩展了启动子研究，对一组拥有相似氨基酸序列的基因进行了重新分类。此外，我们还利用聚合酶链式反应(PCR)产生的单链DNA探针开发了一种独特而灵敏的原位杂交系统，并利用该技术研究了每个BMP基因是如何受到包括Hox基因在内的转录因子的调控的。在本研究期间，我们特别鉴定了BMP-5和BMP-6基因启动子在正常和病理条件下的前列腺癌，发现BMP-6基因受Sp1转录因子结合位点周围的CpG甲基化的表观遗传调控。另一方面，BMP-5缺乏CpG富集区，受Hox基因识别的重复AttA基序调控。作为一项规则，我们进行了我们的研究，以便分子生物学测试总是与形态分析相结合。我们在各种国际和国内会议上发表了我们的数据，并在学术期刊上发表了许多科学论文。

Bone morphogenetic proteins (BMPs), first identified as factors inducing heterogeneous bone and cartilaginous tissues, are now known to play important roles in organogenesis during embryonic development. The amino acids sequences of BMPs are also known to be well conserved from Drosophila to mammalian. So far, BMPs are classified into several subgroups according to their amino acids holomogy. Little is known, however, about the physiologic role and functional sharing of each BMP.In this study, we tried to characterize each BMP's functional sharing by analyzing promoter region of the each BMP gene regardless of the recent classification system based on amino acid homology. The characterization of 5'-flanking region, a promoter structure, is now mostly for the study of the regulation mechanism of gene expression of the specific target gene. By inclusive molecular cloning of the promoter area, we extended the promoter study to 're'-classify a group of genes sharing similar amino acid sequences. Furthermore, we have developed an unique and sensitive in situ hybridization system using a single-stranded DNA probes yielded by polymerase chain reaction (PCR), and used that technique to study how each BMP gene was regulated by transcriptional factors including Hox genes. During this research period, we especially characterized BMP-5 and BMP-6 gene promoters both in normal and pathologic condition as prostate cancer, and found that BMP-6 gene was epigenetically regulated by CpG methylation around the Sp1 transcriptional factor binding sites. On the other hand, BMP-5 lacks such CpG rich area, and regulated by repeated ATTA motifs recognized by Hox genes. As a rule, we performed our studies so that molecular biological assay always went together with morphological analysis. We have presented our data at various international as well as domestic meetings, and publish a lot of scientific papers for academic journals.