基因组的三维(3D)组织对于染色质调节很重要。这种组织是非随机的,似乎与染色质状态和支架蛋白密切相关或受其调节。为了了解特定的DNA和染色质元件如何有助于基因组的功能组织,我们开发了一种新工具-标记的染色体插入位点(TCIS)系统,以鉴定和研究驱动核分隔的最小DNA序列,并应用该系统专门研究顺式元件在将DNA靶向核层中的作用.TCIS系统允许Cre重组酶介导的任何DNA片段定点整合到用lacO阵列标记的基因座中,从而使功能分子研究和位置分析改变的基因座。该系统可用于研究靶向核外围(或其他核区室)的最小DNA序列,让研究人员了解全基因组的结果是如何获得的,例如,通过DNA腺嘌呤甲基转移酶鉴定,染色体构象捕获(HiC),或相关方法,在单细胞水平连接到DNA和染色体的实际组织。最后,TCIS允许测试特定蛋白质在染色质重组中的作用,并确定核环境的变化如何影响单个基因座的染色质状态和基因调控。
The three-dimensional (3D) organization of the genome is important for chromatin regulation. This organization is nonrandom and appears to be tightly correlated with or regulated by chromatin state and scaffolding proteins. To understand how specific DNA and chromatin elements contribute to the functional organization of the genome, we developed a new tool-the tagged chromosomal insertion site (TCIS) system-to identify and
study minimal DNA sequences that drive nuclear compartmentalization and applied this system to specifically
study the role of cis elements in targeting DNA to the nuclear lamina. The TCIS system allows Cre-recombinase-mediated site-directed integration of any DNA fragment into a locus tagged with lacO arrays, thus enabling both functional molecular studies and positional analysis of the altered locus. This system can be used to
study the minimal DNA sequences that target the nuclear periphery (or other nuclear compartments), allowing researchers to understand how genome-wide results obtained, for example, by DNA adenine methyltransferase identification, chromosome conformation capture (HiC), or related methods, connect to the actual organization of DNA and chromosomes at the single-cell level. Finally, TCIS allows one to test roles for specific proteins in chromatin reorganization and to determine how changes in nuclear environment affect chromatin state and gene regulation at a single locus.