Systematic dissection of regulatory motifs in 2000 predicted human enhancers using a massively parallel reporter assay
Systematic dissection of regulatory motifs in 2000 predicted human enhancers using a massively parallel reporter assay
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DOI：
10.1101/gr.144899.112
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发表时间：
2013-05-01
期刊：
影响因子：
7
通讯作者：
Kellis, Manolis
中科院分区：
文献类型：
--
作者：
Kheradpour, Pouya;Ernst, Jason;Kellis, Manolis
全基因组染色质注释允许跨多种人类细胞类型的推定调控元件的映射。然而，他们的实验解剖定向调控基序破坏仍然是不可行的，在基因组规模。在这里，我们使用大规模平行报告分析（MPRA）来测量由145 bp的DNA片段集中在进化上保守的调节基序的增强子染色质状态的情况下诱导的转录水平。我们选择了五个预测的激活子（HNF 1、HNF 4、FOXA、加塔、NFE 2L 2）和两个预测的抑制子（GFI 1、ZFP 161），并测量报告基因在红白血病（K562）和肝癌（HepG 2）细胞系中的表达。我们测试了2104个野生型序列和3314个含有靶向基序破坏的工程增强子变体，每个使用10个条形码标签和两个重复。由此产生的数据有力地证实了增强子染色质状态的增强子活性和细胞类型特异性，145 bp片段重演两者的能力，增强子功能中调控基序的必要作用，以及激活子和阻遏物基序的互补作用。我们发现了统计学上可靠的证据，即（1）破坏预测的激活基序消除了增强子功能，而沉默或基序改善的变化保持了增强子活性;（2）进化保守性，核小体排斥，其他因子的结合和基序匹配的强度预测了增强子活性;（3）在增强子通常无活性的细胞系中，扰乱阻遏物基序导致异常报告基因表达。我们的研究结果表明，通过系统的大规模操作来破译顺式调控元件的一般策略，并提供了数千个构建体的定量增强子活性测量，这些构建体可以被挖掘以开发基因表达的预测模型。
Genome-wide chromatin annotations have permitted the mapping of putative regulatory elements across multiple human cell types. However, their experimental dissection by directed regulatory motif disruption has remained unfeasible at the genome scale. Here, we use a massively parallel reporter assay (MPRA) to measure the transcriptional levels induced by 145-bp DNA segments centered on evolutionarily conserved regulatory motif instances within enhancer chromatin states. We select five predicted activators (HNF1, HNF4, FOXA, GATA, NFE2L2) and two predicted repressors (GFI1, ZFP161) and measure reporter expression in erythroleukemia (K562) and liver carcinoma (HepG2) cell lines. We test 2104 wild-type sequences and 3314 engineered enhancer variants containing targeted motif disruptions, each using 10 barcode tags and two replicates. The resulting data strongly confirm the enhancer activity and cell-type specificity of enhancer chromatin states, the ability of 145-bp segments to recapitulate both, the necessary role of regulatory motifs in enhancer function, and the complementary roles of activator and repressor motifs. We find statistically robust evidence that (1) disrupting the predicted activator motifs abolishes enhancer function, while silent or motif-improving changes maintain enhancer activity; (2) evolutionary conservation, nucleosome exclusion, binding of other factors, and strength of the motif match are predictive of enhancer activity; (3) scrambling repressor motifs leads to aberrant reporter expression in cell lines where the enhancers are usually inactive. Our results suggest a general strategy for deciphering cis-regulatory elements by systematic large-scale manipulation and provide quantitative enhancer activity measurements across thousands of constructs that can be mined to develop predictive models of gene expression.