Roles of regulation of DNA replication in genomic imprinting

染色质的表观遗传修饰在基因组印迹中起着重要作用。在这项研究中，首先开发了DNA复制分析的基本技术。使用新的和以前的技术，染色质的表观遗传修饰对DNA复制的影响进行了检查。主要研究结果如下：1.采用低渗位移法将修饰核苷酸导入细胞，并进行了DNA复制分析。与珠加载方法相反，这不仅可用于贴壁细胞，而且可用于漂浮细胞。2.建立了一种在显微镜盖玻片上精确、均匀地延伸DNA分子的动态分子梳方法，并将其应用于DNA复制分析。同时还建立了从单个细胞中制备半延伸DNA纤维的方法。使用此方法， 关于我们 3. DNA纤维的直接可视化显示，当复制叉穿过R/G染色体带边界和着丝粒周围异染色质时，复制叉移动的速率在S期的早中期典型地降低。研究还表明，组蛋白超乙酰化与染色质结构的变化、复制时间（起点激发）和着丝粒异染色质区域的分叉进展密切相关。S期检查点因子被认为是核DNA损伤后抑制起始放电和稳定复制叉的重要因素。4.核DNA通常分别在复制前和复制后结合和释放到核基质中，尽管MAR区域始终通过S期与其结合。5.通过以下方法促进了研究DNA复制在哺乳动物发育的表观遗传学中的作用的基础研究：印迹结构域的复制时间分析，从ES细胞延伸的复制DNA纤维的可视化，等等。

Epigenetic modifications of chromatin play important roles in genomic imprinting. In this research, basic techniques for the analysis of DNA replication were developed first. Using new and previous techniques, effects of epigenetic modifications of chromatin on DNA replication were examined. The results obtained are listed as follows :1.A hypotonic shift method to introduce modified nucleotides into cells was applied for the analysis of DNA replication. In contrast to the beads-loading method, this can be used not only for adherent cells but for floating cells. Other advantages including efficient and homogeneous incorporation of modified nucleotides were also demonstrated.2.A dynamic molecular combing method that extends DNA molecules precisely and uniformly on a microscope cover slip was established, and applied to analysis of DNA replication. A preparation method of semi-extended DNA fibers from individual cells was also developed. Using this method, asynchronous replication between … More parental alleles in imprinted regions was demonstrated.3.Direct visualization of DNA fibers showed that the rate of replication fork movement typically decreases in the early-mid S phase when the replication fork proceeds through the R/G chromosomal band boundary and pericentromeric heterochromatin. It was also shown that histone hyperacetylation is tightly associated with changes in chromatin structure, replication timing (origin firing) and fork progression of centromeric heterochromatin regions. S phase checkpoint factors were suggested to be responsible for repression of origin firing and stabilization of replication forks when nuclear DNAs were damaged.4.Nuclear DNAs generally bind and release to the nuclear matrix just before and after their replication, respectively, although MAR regions consistently bind to it through S phase.5.Basic researches to examine roles of DNA replication in epigenetics in mammalian development were promoted through replication timing analysis of imprinted domains, visualization of replicating DNA fibers extended from ES cells, and so on. Less