Epigenetic control of mammalian retrotransposons

我的团队已经开发了几个实验系统来研究哺乳动物反转录转座子的表观遗传控制。利用标记的L1反转录转座子，我们正在研究组织培养细胞中通过逆转录转座依赖机制新插入的L1报告基因的杂化和沉默机制。我们还研究了人类和小鼠基因组中存在的天然L1反转录转座子的表观遗传控制，以及这种控制与L1在不同细胞中表达的关系。我们的实验利用了包括发夹状亚硫酸盐测序、染色质免疫沉淀和短抑制RNA等技术对两条DNA链进行DNA甲基化分析。这些实验表明，DNA甲基化本身并不能解释体细胞组织中L1转录的异常强烈的负调控。我们成功地对几个全序列基因表达分析(SAGE)长标签文库进行了测序和分析，以表征具有表观遗传控制改变的体细胞中的整体转录水平，包括对应于重复和转座元件的转录水平。到目前为止，已建成的文库包括大量以非常高的比例差异表达的转录本。差异表达的转录本已经通过包括Northern印迹和定量聚合酶链式反应(QPCR)实验在内的独立技术进行了鉴定和验证。我们现在正在进一步研究基因表达变化与表观遗传控制变化之间的关系。

My group has developed several experimental systems to study epigenetic control of mammalian retrotransposons. Using a marked L1 retrotransposon, we are studying the mechanism of variegation and silencing of L1 reporters that are newly inserted via a retrotransposition-dependent mechanism in tissue culture cells. We have also studied epigenetic control of native L1 retrotransposons pre-existing in the human and mouse genomes, and the relationship between such control and L1 expression in various cells. Our experiments have utilized techiques includingDNA methylation analysis of both DNA strands by hairpin bisulfite sequencing, chromatin immunoprecipitation, and short inhibitory RNAs. These experiments, as a test of the "genome defense model", have shown that DNA methylation does not by itself account for exceptionally strong negative regulation of L1 transcription in somatic tissues.We have successfully sequenced and analyzed several full Serial Analysis of Gene Expression (SAGE) long-tag libraries to characterize global transcript levels, including those corresponding to repetitive and transposable elements, in somatic cells with altered epigenetic controls. The libraries completed to date include numerous transcripts that are differentially expressed at very high ratios. Differentially expressed transcripts have been identified and validated by independent techniques including Northern blotting and quantitative polymerase chain reaction (qPCR) experiments. We are now further characterizing how altered expression may be related to changes in epigenetic control.