Assembly of additional heterochromatin distinct from centromere-kinetochore chromatin is required for de novo formation of human artificial chromosome
Assembly of additional heterochromatin distinct from centromere-kinetochore chromatin is required for de novo formation of human artificial chromosome
复制标题
DOI：
10.1242/jcs.02702
复制
发表时间：
2005-12-15
影响因子：
4
通讯作者：
Masumoto, H
中科院分区：
文献类型：
--
作者：
Nakashima, H;Nakano, M;Masumoto, H
α-卫星（类α）DNA对于人类培养细胞中人类人工染色体（HACs）的从头形成是必需的。为了研究着丝粒、转录许可和非许可染色质组装与从头形成HAC之间的关系，我们构建了基于细菌人工染色体（BAC）的线性HAC载体，其左臂被βgeo编码基因占据，该基因带有或不带有功能性启动子，右臂上还有一个共同的标记基因。尽管在HT1080细胞中从左臂无启动子构建体的载体成功产生了HAC，但我们未能从左臂有功能性启动子的载体产生稳定的HAC。尽管在HAC形成方面失败了，但着丝粒成分（CENP - A、CENP - B和CENP - C）在整合位点组装，这与载体臂上两个标记基因的转录活性状态相关。然而，在稳定的HAC上，染色质免疫沉淀分析表明，HP1α和三甲基组蛋白H3 - K9在不转录的左臂富集。载体臂上的转录活性状态与引入的BAC DNA上异染色质的形成不相容，这表明异染色质的表观遗传组装不同于着丝粒染色质组装，并且是建立稳定人工染色体所必需的。
Alpha-satellite (alphoid) DNA is necessary for de novo formation of human artificial chromosomes (HACs) in human cultured cells. To investigate the relationship among centromeric, transcriptionally permissive and nonpermissive chromatin assemblies on de novo HAC formation, we constructed bacterial artificial chromosome (BAC)-based linear HAC vectors whose left vector arms are occupied by beta geo coding genes with or without a functional promoter in addition to a common marker gene on the right arm. Although HACs were successfully generated from the vectors with promoter-less constructs on the left arm in HT1080 cells, we failed to generate a stable HAC from the vectors with a functional promoter on the left arm. Despite this failure in HAC formation, centromere components (CENP-A, CENP-B and CENP-C) assembled at the integration sites correlating with a transcriptionally active state of both marker genes on the vector arms. However, on the stable HAC, chromatin immunoprecipitation analysis showed that HP1 alpha and trimethyl histone H3-K9 were enriched at the nontranscribing left vector arm. A transcriptionally active state on both vector arms is not compatible with heterochromatin formation on the introduced BAC DNA, suggesting that epigenetic assembly of heterochromatin is distinct from centromere chromatin assembly and is required for the establishment of a stable artificial chromosome.