甲基化全方位解读

readok2009

February 17, 2008
A Widescreen View of Methylation

If the wrong gene is turned on at the wrong time, it can wreak havoc in the cell. To prevent this from happening, organisms rely on DNA methylation to keep unneeded genes turned off. Despite its importance, however, methylation has remained enigmatic — largely because researchers have been forced to examine individual genes one at a time, a myopic and laborious process.

Now, novel molecular biology techniques and software developed by Howard Hughes Medical Institute (HHMI) investigator Steven Jacobsen and colleagues will give scientists the tools they need to identify large-scale methylation patterns in complete genomes. The team published their findings February 17, 2008, in an advanced online publication of the journal Nature.

“There’s lots of evidence that when genes’ methylation patterns are not properly maintained, that is a major cause of cancer. So if we can learn enough about these methylation mechanisms, we may some day learn to manipulate them and treat cancer.”
Steven E. Jacobsen

During DNA methylation, small molecules called methyl groups are added to specific sites on DNA. The methyl groups are attached only to specific cytosine (C) bases — one of the four building blocks of DNA. The methyl group “serves as a beacon, saying that `that stretch of DNA should be silenced,'” explained Jacobsen, whose lab is at the University of California, Los Angeles (UCLA).

Jacobsen has been working to understand how gene silencing happens in the plant Arabidopsis thaliana, a common model organism. His lab has created mutant strains of Arabidopsis that have been useful in observing just how cells attach the chemical signals in exactly the right places.

Methylation is vital to most organisms - even the small Arabidopsis genome contains 13 million methylated cytosines. But Jacobsen says that researchers have struggled to see its exact footprint on the genome. To find methylated genes, researchers usually turn to a technique called bisulphite sequencing, which chemically changes normal cytosine to thymidine (T; another DNA base), while leaving methylated cytosines unchanged. Once this step is complete, the modified DNA can be sequenced.

“In the past we have had to use very labor-intensive, painstaking techniques and look at one gene at a time,” Jacobsen said. This restricted researchers to looking at only a few genes in a genome—stifling their ability to uncover large-scale patterns of methylation.

In the experiments reported in Nature, Jacobsen's group collaborated with computational biologists Matteo Pellegrini and Shawn Cokus, as well as researchers from the biotechnology companies Illumina and New England BioLabs to create new molecular biology methods, algorithms, and software to analyze bisulphite sequences. The techniques allowed the team to correctly and rapidly identify more than 90 percent of the methylated cytosines across the entire genome of Arabidopsis.

Their analysis confirmed methlyation patterns that other researchers' experiments had hinted at, and offered clues into how the enzymes that methylate DNA do their work. For example, they found that among pairs of cytosines, those that were 10 bases apart had the best chance of both being methylated. The reason? “Ten bases is exactly the length of the turn of the double helix of DNA,” Jacobsen said. Their observation suggests that methylating enzymes may travel along one face of the DNA molecule, placing multiple methyl groups at once.

The team also found that the chance of methylation increased at intervals of 167 bases. This matches the spacing between the histone proteins that package DNA,” Jacobsen noted. “We think it's because the enzymes are being pulled in by the histones, and methylating the DNA right nearby.”

According to Jacobsen, his team's findings improve understanding of how cells control gene expression, and might one day find use in medicine. “There's lots of evidence that when genes' methylation patterns are not properly maintained, that is a major cause of cancer,” he said. “So if we can learn enough about these [methylation] mechanisms, we may some day learn to manipulate them” and treat cancer.

Jacobsen said that the Arabidopsis study's success validates their new tools. “Now we're looking at other organisms to see how widely conserved these [patterns] are,” he said. The team is looking for help to search as many genomes as possible; they've posted the source code and additional information about their experiments on their website, http://epigenomics.mcdb.ucla.edu/BS-Seq/. They don't know quite what they'll find, Jacobsen said. “We don't have this kind of data for any other organism yet.”

http://www.hhmi.org/news/jacobsen20080228.html
如果不适当的基因不适时地被启动，可能导致细胞内大破坏。为避免此类事件发生，机体通过DNA甲基化使不需要的基因沉默。尽管它是如此重要，但甲基化至今仍是个难题－主要因为研究者必须逐个检测个体的每个基因，这是一个繁琐且费劲的过程。现在，来自霍德华休斯医学院(HHMI)的研究者史蒂文-雅克布森及同事开发了新颖的分子生物学技术和软件，将给科学家带来他们需要的全基因组甲基化状态检测工具。这一研究成果发表在2008年2月17日《自然》杂志在线版上。“有很多证据表明，基因甲基化状态不正常是肿瘤发生的一个主要原因。因此，如果我们对这些甲基化机制了解的足够透彻，也许有朝一日可以操纵他们、治疗肿瘤。”史蒂文-雅克布森说。DNA甲基化过程中，小分子（甲基团）被添加到DNA的特定位点。这些甲基团只粘附于特定胞嘧啶碱基－DNA四个组成成分之一。甲基团“充当一个指示标，告知机体‘那段DNA应该沉默掉，’”雅克布森解释，它的实验室在洛杉矶加州大学(UCLA)。雅克布森一直致力于常见模式生物－拟南芥怎样基因沉默的研究。他的实验室已经培育出拟南芥突变株，这对于观察细胞怎样将化学信号分子精确定位非常有用。甲基化作用对绝大数有机体至关重要－就算是微小的拟南芥基因组也包含了1300万甲基化胞嘧啶碱基。但是雅克布森说，研究者们已经致力于基因组甲基化精确足迹的艰难寻找。研究者通常借助于亚硫酸氢盐测序技术检测甲基化的基因，它通过化学反应使正常胞嘧啶转变为胸腺嘧啶（T：另一种DNA碱基），而对甲基化的胞嘧啶无作用。一旦这一步完成，被修饰的DNA就可以被测序了。“在过去，我们只能用让人非常辛苦的劳力密集型技术，并且一次只能检测一个基因，”雅克布森说。这让研究者们只能就一个基因组的少数几个基因开展研究－为了寻找大规模的甲基化状态，他们的工作能力被枯燥地消磨掉。在《自然》杂志里如是报道，雅克布森的团队与计算生物学家Matteo Pellegrini ＆ Shawn Cokus合作，还有来自Illumina生物技术公司和新英格兰生物实验室的研究者们，建立了新的分子生物学方法、算法和软件，用于解析亚硫酸氢盐测序。这项技术让研究小组正确快速地辨别多于90％的拟南芥全基因组甲基化胞嘧啶。他们的分析证实了其他研究者实验提示的甲基化状态，并且为酶怎样甲基化DNA从而发挥他们的作用提供线索。例如，他们发现在成对的胞嘧啶中，那些相隔10个碱基的胞嘧啶均被甲基化的概率最大。原因何在？“10个碱基正好是DNA双螺旋一圈的长度，”雅克布森说。他们的研究提示，甲基化酶可能延着DNA分子的一面移动，一次性添加多个甲基团。研究小组同时发现，间隔167碱基时甲基化的概率增加了。这与包装着DNA的组蛋白间距一致，”雅克布森指出。“我们认为，那是因为酶被嵌进了组蛋白之间，且就近甲基化DNA。”根据雅克布森（的评论），他团队的发现深化了对细胞怎样调控基因表达的理解，并且可能有朝一日应用于医学。“有很多证据表明，基因甲基化状态不正常是肿瘤发生的一个主要原因，”他说。“因此，如果我们对这些甲基化机制了解的足够透彻，也许有朝一日可以操纵他们”、治疗肿瘤。雅克布森说拟南芥研究的成功验证了他们的新工具。“现在我们正在检测其他有机体，探究这些保守结构到底有多普遍，”他说。团队正在寻求帮助以期搜索尽可能多的基因组；他们已经在他们的网站上公布了源代码和实验的相关信息，http://epigenomics.mcdb.ucla.edu/BS-Seq/。他们对自己的发现不是很理解，雅克布森说。“我们还没有其他有机体的此类数据。”