A genome-integrated massively parallel reporter assay reveals DNA sequence determinants of cis-regulatory activity in neural cells.
A genome-integrated massively parallel reporter assay reveals DNA sequence determinants of cis-regulatory activity in neural cells.
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DOI：
10.1093/nar/gkw942
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发表时间：
2017-02-28
影响因子：
14.9
通讯作者：
Cohen BA
中科院分区：
文献类型：
--
作者：
Maricque BB;Dougherty JD;Cohen BA
最近大规模的基因组学研究对协调全基因组表达模式的顺式调控序列进行了表征，并产生了令人印象深刻的假定调控元件目录。这些序列中的大多数都没有经过功能测试，而且我们对非编码基因组的有限理解使我们无法预测哪些序列是真正的顺式调控元件。最近，大规模平行报告基因测定（MPRAs）已被用于测量几种生物学背景下假定的顺式调控序列的活性，每种序列都具有特定的优势和明显的局限性。我们开发了LV-MPRA，一种新型的基于慢病毒的大规模平行报告基因检测，用于研究基因组整合调控元件在任何哺乳动物细胞类型中的功能；因此，可以将mpra应用于与生物学更相关的情况。我们测量了U87胶质母细胞瘤细胞和人类神经祖细胞（hNPCs）中2600个序列的活性，并探索了调控活性如何在DNA序列中编码。我们证明LV-MPRA可以应用于估计局部DNA序列和区域染色质对调控活性的影响。我们的数据显示，主要的DNA序列特征，如GC含量和二核苷酸组成，在全染色体背景下准确区分高活性序列和低活性序列，并且还可能与不同的转录因子结合位点结合，以确定细胞类型特异性。我们得出结论，LV-MPRA将成为鉴定顺式调控元件和促进对非编码基因组如何编码信息的新认识的重要工具。
Recent large-scale genomics efforts to characterize the cis-regulatory sequences that orchestrate genome-wide expression patterns have produced impressive catalogues of putative regulatory elements. Most of these sequences have not been functionally tested, and our limited understanding of the non-coding genome prevents us from predicting which sequences are bona fide cis-regulatory elements. Recently, massively parallel reporter assays (MPRAs) have been deployed to measure the activity of putative cis-regulatory sequences in several biological contexts, each with specific advantages and distinct limitations. We developed LV-MPRA, a novel lentiviral-based, massively parallel reporter gene assay, to study the function of genome-integrated regulatory elements in any mammalian cell type; thus, making it possible to apply MPRAs in more biologically relevant contexts. We measured the activity of 2,600 sequences in U87 glioblastoma cells and human neural progenitor cells (hNPCs) and explored how regulatory activity is encoded in DNA sequence. We demonstrate that LV-MPRA can be applied to estimate the effects of local DNA sequence and regional chromatin on regulatory activity. Our data reveal that primary DNA sequence features, such as GC content and dinucleotide composition, accurately distinguish sequences with high activity from sequences with low activity in a full chromosomal context, and may also function in combination with different transcription factor binding sites to determine cell type specificity. We conclude that LV-MPRA will be an important tool for identifying cis-regulatory elements and stimulating new understanding about how the non-coding genome encodes information.