Targeted disruption of the MARs in the human imprinted genes cluster

在哺乳动物中，大多数印迹基因聚集在大的基因组域中，并且经常共享共同的调控元件。基因表达的调控被认为是一个多步骤的过程，涉及染色质结构的变化，通过位点特异性事件，如DNA和组蛋白的甲基化或乙酰化。基质附着区（MARs）是基因组中附着在核基质上的DNA元件。MARs位于几个大基因的内含子中，也作为转录单元的边界，提示MARs在染色质结构域下调节基因的转录。它可能是控制基因组印记等大碱基范围内的长期表观遗传效应的一个因素。为了证明这一假设，我们研究了火星在印迹域的功能。我们利用重组鸡DT40细胞，在人11p15.5染色体上靶向缺失IGF2和LIT1中的MARs，构建了修饰的人染色体。此外，我们将修饰的人类染色体转移到中国仓鼠卵巢（CHO）细胞中，分析该印迹簇靶向改变的影响。印迹非编码RNA LIT1是kcnq10t1的产物，参与了人类染色体11p15.5上一个印迹簇的顺式限制性沉默。虽然该基因座是印迹中心（imprinting center， IC），但LIT1基因产物的功能尚不清楚。在本研究中，RNA原位杂交提供的证据表明，LIT1 RNA稳定地定位于LIT1区域，并在印迹结构域的转录沉默中发挥重要作用。因此，为了更好地理解新的表观遗传基因调控，包括印迹基因，未来的研究重点将放在染色质与核基质之间的相互作用上。

In mammals, most imprinted genes are clustered into large genomic domains and often share common regulatory elements. Regulation of gene expression is thought to be a multistep process involving changes in chromatin structure by site-specific events such as the methylation or the acethylation on DNA and histones. Matrix attachment regions(MARs) are DNA elements for the genome that attach to the nuclear matrix. MARs are located in the intron of several large genes and also act as the borders of transcription unit, suggesting that MARs play a role in modulating transcription of the genes under the chromatin domain. It may contribute to a factor that control long-range epigenetic effects on a megabase scale such as genomic imprinting.To demonstrate this hypothesis, we investigated the function of MARs in imprinted domain. Here we created modified human chromosome carrying the targeted deletion of MARs in IGF2 and LIT1 on human chromosome 11p15.5 using recombination-proficient chicken DT40 cells. Furthermore, we transferred the modified human chromosome into chinese hamster ovary(CHO) cells to analyze the effect of the targeted alterations at this imprinted cluster.The imprinted noncoding RNA LIT1, a product of the KCNQ1OT1, is involved in cis-limited silencing within an imprinted cluster on human chromosome 11p15.5. Although the locus serves as an imprinting center (IC), the function of the LIT1 gene product is unclear. In this study, RNA in situ hybridization provides evidence suggesting that the LIT1 RNA stably localizes to the LIT1 region and plays an important role in transcriptional silencing of the imprinting domain.Thus, it will be important to focus future study on the interactions between chromatin and the nuclear matrix in order to better understand new epigenetic gene regulation, including imprinted genes.