Epigenetic control of mammalian retrotransposons

我的团队已经确定了数千种基因组插入/删除（indel）二态性，这些二态性区分了不同的近交小鼠品系。我们发现大多数较大的二态索引是由于最近的反转录转座子，特别是L1 （LINE）反转录转座子。所查询的所有候选二态性已通过PCR检测验证。二态转座子具有真正的近期整合事件的所有典型特征，包括靶位点复制、典型靶特异性、长多聚(A)尾、基因内含子内的优先定位和基因定向反义以及长度分布。我们还发现了许多新的转录结构，直接归因于这些最近整合的转座子。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元件在培养的癌细胞系中在几周内积极移动。这些结果表明，转座子可以显著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，不成比例的二态内源性转座子位于或靠近脑表达基因。许多新的转录结构，直接归因于最近的转座子整合物，在大脑和/或睾丸中特异性表达。大多数内源性转座子仅在某些组织中表达，通常在发育早期，如脑和睾丸。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逆转录转座子，我们研究了组织培养细胞中通过逆转录转座子依赖机制新插入的L1报告子的变异和沉默机制。我们发现DNA甲基化在这些新整合物的沉默中并不起主要作用，而是动态调节的组蛋白乙酰化和去乙酰化与可变的报告基因表达相关。我们修改了一种程序，将报告子与其他元件（如绝缘子、反转录转座子等）的片段一起引入定义的遗传位点，即重组介导的盒式交换。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，以表征具有改变表观遗传控制的体细胞中的全局转录物水平，包括那些与重复和转座元件相对应的转录物水平。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。通过Northern blotting和定量聚合酶链反应（qPCR）实验等独立技术，已经鉴定并验证了许多差异表达转录物，包括几个基因家族或途径的成员，如癌癌基因、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描述改变的表达如何与表观遗传控制的变化相关。

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.