Pesenti Elisa, Kouprina Natalay, Liskovykh Mikhail, Aurich-Costa Joan, Larionov Vladimir, Masumoto Hiroshi, Earnshaw William C, Molina Oscar
Wellcome Trust Centre for Cell Biology, University of Edinburgh , Edinburgh EH9 3QR, United Kingdom.
Genome Structure and Function Group, Developmental Therapeutics Branch, National Cancer Institute , National Institutes of Health , Bethesda , Maryland 20892 , United States.
ACS Synth Biol. 2018 Apr 20;7(4):1116-1130. doi: 10.1021/acssynbio.8b00018. Epub 2018 Mar 29.
It is generally accepted that chromatin containing the histone H3 variant CENP-A is an epigenetic mark maintaining centromere identity. However, the pathways leading to the formation and maintenance of centromere chromatin remain poorly characterized due to difficulties of analysis of centromeric repeats in native chromosomes. To address this problem, in our previous studies we generated a human artificial chromosome (HAC) whose centromere contains a synthetic alpha-satellite (alphoid) DNA array containing the tetracycline operator, the alphoid-HAC. The presence of tetO sequences allows the specific targeting of the centromeric region in the HAC with different chromatin modifiers fused to the tetracycline repressor. The alphoid-HAC has been extensively used to investigate protein interactions within the kinetochore and to define the epigenetic signature of centromeric chromatin to maintain a functional kinetochore. In this study, we developed a novel synthetic HAC containing two alphoid DNA arrays with different targeting sequences, tetO, lacO and gal4, the alphoid-HAC. This new HAC can be used for detailed epigenetic engineering studies because its kinetochore can be simultaneously or independently targeted by different chromatin modifiers and other fusion proteins.
一般认为，含有组蛋白H3变体CENP - A的染色质是维持着丝粒身份的表观遗传标记。然而，由于分析天然染色体中着丝粒重复序列存在困难，导致着丝粒染色质形成和维持的途径仍未得到充分表征。为了解决这个问题，在我们之前的研究中，我们构建了一种人类人工染色体（HAC），其着丝粒包含一个含有四环素操纵子的合成α - 卫星（alphoid）DNA阵列，即alphoid - HAC。tetO序列的存在使得可以用与四环素阻遏物融合的不同染色质修饰剂特异性靶向HAC中的着丝粒区域。alphoid - HAC已被广泛用于研究动粒内的蛋白质相互作用，并确定着丝粒染色质的表观遗传特征以维持功能性动粒。在本研究中，我们构建了一种新型合成HAC，它含有两个具有不同靶向序列tetO、lacO和gal4的alphoid DNA阵列，即alphoid - HAC。这种新的HAC可用于详细的表观遗传工程研究，因为其动粒可以同时或独立地被不同的染色质修饰剂和其他融合蛋白靶向。
