Investigating the role of Retrotransposon muERV-L reactivation in Tumorigenesis

 描述（由申请方提供）：研究逆转录转座子muERV-L再激活在肿瘤发生中的作用。背景：在整个进化过程中，古老的外来核酸序列已经感染并传播到几乎所有生物的基因组中。这些寄生元件移动以重新排列它们的位置并在整个宿主基因组中繁殖。42%的人类基因组起源于一类称为反转录转座子的移动的元件，并且已经进化出复杂的机制来抑制异常再激活的潜在致突变作用。虽然非常有效地沉默反转录转座子，特定的反转录转座子家族的发育和组织特异性表达模式表明，这些元素已被其宿主基因组驯化。 初步结果表明，在不存在肿瘤抑制因子p53（小鼠逆转录转座子的古老类别）的情况下诱导多能干细胞期间，muERV-L被去抑制，而几乎所有其他逆转录转座子沉默保持完整。这种发生在数千个基因座上的再激活，伴随着与muERV-L基因座特异性相邻的基因活性的显著失调。这些影响是否有助于或p53丢失的增强致瘤性的后果是本调查的重点。假设：我们的初步研究表明，在诱导缺乏肿瘤抑制基因p53的多能干细胞后，逆转录转座子muERV-L被重新激活，对邻近基因的活性产生显著影响。因此，我假设p53介导muERV-L逆转录转座子沉默在肿瘤的发展。具体目的：（1）通过muERV-L破坏来挽救p53缺陷型iPS细胞中改变的基因表达。(2)表征体内癌症发展中的异常反转录转座子活性。(3)研究p53抑制特定反转录转座子的机制。研究设计：加州大学伯克利分校开创的新型CRISPR基因组编辑技术将用于破坏特定的muERV-L基因座，以评估潜在顺式调控效应的变化。接下来，将使用两种不同的肺癌小鼠模型来研究muERV-L再活化对肿瘤形成的体内作用。p53缺陷细胞系和小鼠模型的遗传，生物化学和分子方法将被用来研究p53下游的表观遗传和转录机制，赋予这种序列特异性调节逆转录转座子沉默。

 DESCRIPTION (provided by applicant): Investigating the role of Retrotransposon muERV-L reactivation in Tumorigenesis. Background: Throughout evolution, ancient foreign nucleic acid sequences have infected and spread throughout the genomes of nearly all organisms. These parasitic elements mobilize in order to rearrange their position and propagate throughout the host genome. 42% of the human genome originates from a class of mobile element called retrotransposons and has evolved elaborate mechanisms to suppress the potentially mutagenic effects of aberrant reactivations. Although exceedingly efficient at silencing retrotransposons, developmental and tissue specific expression patterns of specific retrotransposon families suggest that these elements have been domesticated by their host genomes. Preliminary results demonstrate that during the induction of pluripotent stem cells in the absence of the tumor suppressor p53, the ancient class of mouse retrotransposon, muERV-L is de-repressed while nearly all other retrotransposon silencing remains intact. This reactivation, occurring at thousands of loci, is accompanied by the significant misregulation of gene activity specifically adjacent to muERV-L loci. Whether these effects contribute to or are a consequence of the enhanced tumorigenicity of p53 loss is the focus of this investigation. Hypothesis: Our preliminary studies indicate that upon induction of pluripotent stem cells lacking the tumor suppressor p53, the retrotransposon muERV-L is reactivated with significant impact on neighboring gene activity. Therefore, I hypothesize that p53 mediates muERV-L retrotransposon silencing during tumor development. Specific Aims: (1) Rescue altered gene expression in p53 deficient iPS cells via muERV-L disruption. (2) Characterize aberrant retrotransposon activity in in vivo cancer development. (3) Investigate the mechanism through which p53 represses specific retrotransposons. Study design: The novel CRISPR genome editing technique pioneered here at UC Berkeley will be used to disrupt specific muERV-L loci to assess changes on potential cis regulatory effects. Next, two distinct mouse models of lung cancer will be used to investigate the in vivo effect of muERV-L reactivation on tumor formation. Genetic, biochemical and molecular approaches on p53 deficient cell lines and mouse models will be used to investigate the epigenetic and transcriptional machinery downstream of p53 that confer this sequence-specific regulation for retrotransposon silencing.