A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity
A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity
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DOI：
10.1101/gr.212092.116
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发表时间：
2017-01-01
期刊：
影响因子：
7
通讯作者：
Shendure, Jay
中科院分区：
文献类型：
--
作者：
Inoue, Fumitaka;Kircher, Martin;Shendure, Jay
可以根据染色质修饰、染色质修饰剂与转录因子和辅因子的结合或染色质可及性来鉴定候选增强子。然而，验证此类候选物作为真正的增强子需要功能表征，通常通过报告基因测定来实现，该测定测试序列是否可以通过最小启动子增加转录报告基因的表达。一个长期存在的担忧是报告基因检测主要在附加体上进行，而附加体被认为缺乏生理染色质。然而，附加体与染色体中的调节序列的顺式调节差异的大小和决定因素仍然几乎完全未知。为了系统地解决这个问题，我们开发并应用了一种新型的基于慢病毒的大规模并行报告基因测定（lentiMPRA），以直接比较 2236 种候选肝脏增强剂在附加型与染色体整合环境中的功能活性。我们发现染色体整合序列的活性与附加体上测定的相同序列的活性有很大不同，而且与 ENCODE 注释的不同子集相关。通过 ENCODE 注释和基于序列的模型，基于染色体的报告基因检测的结果也更具重现性和更强的可预测性。通过结合染色质注释和序列信息的线性模型，我们实现了 0.362 的 Pearson R-2，用于预测染色体整合报告基因检测的结果。这种预测水平比单独使用染色质注释或序列信息更好，并且也优于附加型检测的预测模型。我们的结果对于顺式调控元件的识别、优先排序和功能验证具有广泛的影响。
Candidate enhancers can be identified on the basis of chromatin modifications, the binding of chromatin modifiers and transcription factors and cofactors, or chromatin accessibility. However, validating such candidates as bona fide enhancers requires functional characterization, typically achieved through reporter assays that test whether a sequence can increase expression of a transcriptional reporter via a minimal promoter. A longstanding concern is that reporter assays are mainly implemented on episomes, which are thought to lack physiological chromatin. However, the magnitude and determinants of differences in cis-regulation for regulatory sequences residing in episomes versus chromosomes remain almost completely unknown. To address this systematically, we developed and applied a novel lentivirus-based massively parallel reporter assay (lentiMPRA) to directly compare the functional activities of 2236 candidate liver enhancers in an episomal versus a chromosomally integrated context. We find that the activities of chromosomally integrated sequences are substantially different from the activities of the identical sequences assayed on episomes, and furthermore are correlated with different subsets of ENCODE annotations. The results of chromosomally based reporter assays are also more reproducible and more strongly predictable by both ENCODE annotations and sequence-based models. With a linear model that combines chromatin annotations and sequence information, we achieve a Pearson's R-2 of 0.362 for predicting the results of chromosomally integrated reporter assays. This level of prediction is better than with either chromatin annotations or sequence information alone and also outperforms predictive models of episomal assays. Our results have broad implications for how cis-regulatory elements are identified, prioritized and functionally validated.