Targeted disruption of the MARs in the human imprinted genes cluster

在哺乳动物中，大多数印记基因聚集在大的基因组结构域中，通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，涉及染色质结构的改变，通过位点特异性事件，如DNA和组蛋白上的甲基化或乙酰化。基质附着区(MARS)是连接到核基质上的基因组的DNA元件。MARS位于几个大基因的内含子上，也是转录单位的边界，这表明MARS在调控染色质结构域下基因的转录方面发挥了作用。为了证明这一假设，我们研究了MARS在印迹结构域中的功能。在这里，我们使用精通重组的鸡DT40细胞创造了携带人染色体11p15.5上IGF2和LIT1中MARS靶向缺失的修饰的人染色体。此外，我们将修饰后的人染色体转移到中国仓鼠卵巢(CHO)细胞中，以分析靶向改变对该印迹簇的影响。印迹非编码RNA LIT1是KCNQ1OT1的产物，参与了人染色体11p15.5印迹簇内的顺式限制性沉默。虽然LIT1基因是一个印迹中心(IC)，但其功能尚不清楚。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，未来重点研究染色质与核基质之间的相互作用，以更好地理解新的表观遗传基因调控，包括印记基因。

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.