2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38701CENP-A, C, and H are constitutive centromeric proteins in the inner plate of kinetochore. CENP-A and CENP-C had been characterized as a centromeric DNA associating protein. The loading of CENP-A onto the centromere is the first step of the centromere identity. The loading of CENP-C onto the centromere is an essential step of forming an active centromere. The CENP-C loading is dependent on the both loading of CENP-A and CENP-H. On the other hand, the CENP-H loading is independent on the loading of CENP-A. The loading of CENP-H onto the centromere is a required step for recruiting the nascent CENP-A to form an active centromere de novo. However, it is not yet known as how CENP-H loads onto the centromere. We hypothesize that the loading of CENP-H is achieved by associating with the centromeric DNA. Therefore, to explore what are the CENP-H associating centromeric DNAs, we had first constructed a mini-library from the chromatin immunoprecipitation using anti-CENP-H serum. In our preliminary results, we had obtained five clones with stronger positive signals by means of protein-DNA binding assay. These five clones could contain the candidate CENP-H associating centromeric DNAs. Therefore, in this grant proposal, we plan to characterize these clones including DNA sequence, ex-vivo protein-DNA binding assay, and the ability of assembling other centromeric proteins in the first year of the study. These results will be expected to elucidate that the loading of CENP-H onto the centromeric chromatin is dependent on a specific DNA sequence. Thus, it will in turn shed more light on the underlying mechanism of kinetochore assembly. Furthermore, we would like to explore the genomic organization of CENP-A and CENP-H associating DNAs. We will screen the BAC clones using the CENP-A and CENP-H associating DNAs as probes and then systematically sequence the screened positive BAC clones in second year of the study. This spatial relationship of genomic organization can further demonstrate how the loading of CENP-C onto the centromeric chromatin is dependent on the loading of CENP-A and CENP-H. Finally, we are very much interested in exploring whether it is sufficiency to de novo form an active centromere when the BAC clone contains both the CENP-A and CENP-H associating DNAs. In the third year, we will introduce the BAC clones with CENP-A and CENP-H associating DNAs into mammalian cells and determine the ability of centromeric proteins assembling on the introduced BAC DNAs. We anticipate that these results will shed more light on the essential elements required to form a functional centromere de novo.著絲點蛋白A、C 和H 是位於著絲點Kinetochore 內層的結構性蛋白。著絲點蛋白A 和C 已知是和著絲點DNA 有連結的蛋白。形成著絲點部位的第一步是由著絲點蛋白A 連結上 著絲點DNA，然而形成有活性著絲點還必需要有著絲點蛋白C 聚集在著絲點上。著絲點蛋 白C 要聚集在著絲點則必須依賴已位於著絲點上的著絲點蛋白A 和H 的結合。除此之外， 新生的著絲點蛋白A 要聚集在著絲點也和著絲點上的著絲點蛋白H 有關。但是，著絲點蛋 白H 本身聚集在著絲點和存在於著絲點上的著絲點蛋白A 是互相獨立的。因此，著絲點蛋 白H 如何連結上著絲點是值得探討的課題。我們假設著絲點蛋白H 是藉由和某種著絲點 DNA 連結而聚集在著絲點上。為了研究著絲點蛋白H 連結的著絲點DNA，我們先前已利 用抗著絲點蛋白H 抗體所抓取的染色質絲(也就是染色質絲免疫沉澱法)建立了一個小型染 色質絲資料庫，叫做CHIP-H。在約有300 個來自CHIP-H 的菌株中，我們先任意挑96 個菌株進行細胞外蛋白質-DNA 鍵結分析，其中有五個菌株的DNA 出現較強的連結訊 號。因此，在本計畫中，第一年我們將針對這五個菌株DNA 進行一系列的特性分析，包括 DNA 定序、基因組特性分析和細胞內著絲點蛋白H 的連結能力分析，確立菌株DNA 是著 絲點蛋白H 連結之特定的著絲點DNA，此項結果將證明著絲點蛋白H 是藉由和特定的著 絲點DNA 連結而聚集在著絲點上。第二年我們將探討著絲點蛋白H 連結之特定的著絲點 DNA 和著絲點蛋白A 連結之特定的著絲點DNA 兩者的基因組關係。我們將利用此一著絲 點蛋白H 連結之特定的著絲點DNA 和已知的著絲點蛋白A 連結之特定的著絲點DNA 當 作探針篩選BAC DNAs，然後將有系統地定序候選的BAC DNAs，此一結果將更進一步 說明著絲點蛋白C 聚集在著絲點上是如何依賴已位於著絲點上的著絲點蛋白A 和H。最 後，我們更有興趣探討著絲點蛋白H 連結之特定的著絲點DNA 和著絲點蛋白A 連結之特 定的著絲點DNA兩者同時存在的BAC DNA在細胞內聚集著絲點蛋白以形成有活性的新生 著絲點的能力，因此我們將把候選的BAC DNAs 轉染到細胞內，然後分析著絲點蛋白聚集 在BAC DNA 上的情形。預期此一結果將闡明重新生成著絲點所需要的必要元素。著絲點蛋白H著絲點DNACENP-Hcentromeric DNAProject