Epigenetic control of mammalian retrotransposons

我的团队已经确定了数千种基因组插入/缺失(“indel”)二态，它们区分了不同的近交系小鼠。我们发现，大多数较大的二态Indels是由于最近的逆转录转座，特别是L1(LINE)反转录转座子的作用。所有被询问的候选二态都已通过聚合酶链式反应分析得到验证。二态转座子具有真正的近期整合事件的所有典型特征，包括靶点重复、典型的靶标特异性、长的Poly(A)尾巴、与基因方向相反的基因内含子内的优先定位以及长度分布。我们还鉴定了许多新的转录本结构，直接归因于这些最近整合的转座子中的一些。我们还开发了一种新的分子技术来鉴定新动员的内源性转座子，并表明人类L1元件在几周内就能在培养的癌细胞系中活跃地移动。这些结果表明，转座子可以戏剧性地实时改变正常哺乳动物的基因组和基因表达谱。在小鼠品系中，数量不成比例的二态内源性转座子位于脑中表达的基因或其附近。许多新的转录本结构直接归因于最近的转座子整合子，在脑和/或睾丸中特异表达。大多数内源性转座子只在某些组织中表达，通常是在发育早期，如大脑和睾丸。此外，我的团队开发了一个基于培养细胞的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，我们研究了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。我们发现DNA甲基化并不是这些新整合子沉默的主要原因，而是动态调节的组蛋白乙酰化和去乙酰化与可变报告表达相关。我们已经修改了一种程序，将记者与绝缘子、反转录转座子等其他元件的片段一起引入到定义的遗传位点，即重组介导盒交换。我们已经成功地对几个完整的基因表达序列分析(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全球转录水平，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。大量差异表达的转录本已经被包括Northern印迹和定量聚合酶链式反应(QPCR)实验在内的独立技术鉴定和验证，包括几个基因家族或通路的成员，如肿瘤睾丸基因、干扰素诱导基因、主要组织相容性复合体(MHC)基因和金属硫蛋白基因簇。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 identified thousands of genomic insertion/deletion ("indel") dimorphisms that distinguish different inbred mouse strains. We found that a majority of larger dimorphic indels is due to recent retrotransposition, particularly by L1 (LINE) retrotransposons. All candidate dimorphisms queried have been validated by a PCR assay. The dimorphic transposons have all the typical features of bona fide recent integration events including target site duplications, canonical target specificity, long poly(A) tails, preferential localization within gene introns antisense to gene orientation, and length distribution. We also identified numerous novel transcript structures, attributable directly to some of these recently integrated transposons. We also developed a new molecular technique to identify newly mobilized endogenous transposons, and showed that human L1 elements actively move in cultured cancer cell lines within a few weeks. These results show that transposons can alter the normal mammalian genome, and gene expression profiles, dramatically and in real time. A disproportionate number of dimorphic endogenous transposons in mouse strains are located in or near genes expressed in brain. Numerous novel transcript structures, attributable directly to recent transposon integrants, are specifically expressed in brain and/or testis. Most endogenous transposons are expressed in only certain tissues, typically early in development, such as brain and testis. Additionally, my group has developed a cultured cell-based experimental system to study epigenetic control of mammalian retrotransposons. Using a marked L1 retrotransposon, we have studied the mechanism of variegation and silencing of L1 reporters that are newly inserted via a retrotransposition-dependent mechanism in tissue culture cells. We found that DNA methylation does not play a major role in silencing of these new integrants, but rather that dynamically regulated histone acetylation and deacetylation are correlated with variable reporter expression. We have modified a procedure to introduce reporters together with fragments of other elements such as insulators, retrotransposons, etc. into defined genetic loci, i.e. recombination-mediated cassette exchange. 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. Numerous differentially expressed transcripts have been identified and validated by independent techniques including Northern blotting and quantitative polymerase chain reaction (qPCR) experiments, including members of several gene families or pathways such as cancer testis genes, interferon-inducibile genes, major histocompatibility complex (MHC) genes, and the metallothionein gene cluster. We are now further characterizing how altered expression may be related to changes in epigenetic control.