Epigenetic control of mammalian retrotransposons

我的小组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。使用一个标记的L1反转录转座子，我们正在研究杂色和沉默的L1报告，是新插入的组织培养细胞中通过一个反转录转座子依赖的机制的机制。我们还研究了在人类和小鼠基因组中预先存在的天然L1反转录转座子的表观遗传控制，以及这种控制和L1在各种细胞中的表达之间的关系。我们的实验利用了包括通过发夹亚硫酸氢盐测序、染色质免疫沉淀和短抑制RNA对两条DNA链进行DNA甲基化分析的技术。这些实验，作为“基因组防御模型”的测试，已经表明DNA甲基化本身并不能解释体细胞组织中L1转录异常强烈的负调控。我们已经成功地测序和分析了几个完整的基因表达系列分析（SAGE）长标签文库，以表征全局转录水平，包括那些对应于重复和转座元件的转录水平，在改变了表观遗传控制的体细胞中。迄今为止完成的文库包括许多以非常高的比率差异表达的转录本。差异表达的转录本已经通过独立的技术鉴定和验证，包括北方印迹和定量聚合酶链反应（qPCR）实验。我们现在正在进一步表征表达改变如何与表观遗传控制的变化相关。

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.