ANALYSIS OF THE GENE PROMOTERS OF HUMAN BONE MORPHOGENETIC PROTEINS

骨形态发生蛋白（BMP）最初被认为是诱导异质骨和软骨组织的因子，现在已知在胚胎发育过程中的器官发生中发挥重要作用。还已知 BMP 的氨基酸序列从果蝇到哺乳动物都非常保守。到目前为止，BMP 根据其氨基酸同源性分为几个亚类。然而，人们对每个 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。启动子结构5'侧翼区的表征目前主要用于研究特定靶基因的基因表达调控机制。通过启动子区域的包容性分子克隆，我们扩展了启动子研究，以“重新”分类一组共享相似氨基酸序列的基因。此外，我们使用聚合酶链式反应（PCR）产生的单链DNA探针开发了一种独特且灵敏的原位杂交系统，并利用该技术研究每个BMP基因如何受到包括Hox基因在内的转录因子的调节。在本研究期间，我们特别对正常和前列腺癌病理状态下的BMP-5和BMP-6基因启动子进行了表征，发现BMP-6基因受到Sp1转录因子结合位点周围CpG甲基化的表观遗传调控。另一方面，BMP-5缺乏这种富含CpG的区域，并受到Hox基因识别的重复ATTA基序的调节。通常，我们进行研究时，分子生物学测定总是与形态学分析结合在一起。我们在各种国际和国内会议上展示了我们的数据，并在学术期刊上发表了大量科学论文。

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.