Dinucleotide tag‐based parallel reporter gene assay method enables efficient identification of regulatory mutations

Abstract

Background:The causal single nucleotide polymorphisms (SNPs) leading to increased cancer predisposition mainly function as gene regulatory elements, the evaluation of which largely relies on the parallel reporter gene assay system. However, the common DNA barcodes used in parallel reporter gene assay systems typically because nucleotide composition bias, and many barcodes must be allocated for each sequence to reduce the bias effect. Main methods and major results:Here, a versatile dinucleotide-tag reporter system (DiR) that enables parallel analysis of regulatory elements with minimized bias based on next-generation sequencing is described. The DiR system is more robust than the classical luciferase assay method, particularly for the investigation of moderate-level regulatory elements. The authors applied the DiR-seq assay in the functional evaluation of SNPs with prostate cancer risk and nominated two and six regulatory SNPs in PC-3 and LNCaP cells, respectively. Conclusions and implications:The DiR system has great potential to advance the functional study of SNPs associated with polygenic disease risks. K E Y W O R D Sdinucleotide-tag reporter system (DiR), gene regulation, prostate cancer, single nucleotide polymorphism (SNP) INTRODUCTIONGenome-wide association studies (GWAS) provide a practical approach for disclosing genetic variations associated with complex diseases. [1] The GWAS Catalog contains annotations for over 5000 SNPs with cancer risk. Over 90% of these single nucleotide polymorphisms (SNPs) are located outside protein-coding regions; thus, annotating their functional impacts is challenging.

Strategies for activity analysis of single nucleotide polymorphisms associated with human diseases

“…Moreover, it has been shown that the DiR system has higher sensitivity than the traditional luciferase system. 67,79 It should not be ignored that DiR-seq also has the same drawbacks as MPRAs and STARR-seq systems. The episomal plasmid libraries free from the chromatin environment may not truly reflect chromatin interactions.…”

“…However, the final reported result is a relative activity, thus it is necessary to ensure that the allele segments are at the same length. There is also a non‐negligible limiting factor, that is, the tag bias can only be reduced but not completely eliminated, and the tag bias may affect the activity analysis of reporter elements, especially the reporter elements with weak native activity, such as SNPs found by GWAS 67 . Meanwhile, each DNA sample matches hundreds of barcode tags in order to reduce the tag bias, resulting in a lower sensitivity compared to the traditional luciferase system 66 …”

“…Arbitrary DNA fragments of interest can be inserted upstream of the promoter of the DiR vector, and the relative activity of the DNA fragment can be obtained through the relative expression of the downstream dinucleotide sequence. The DiR‐seq was first developed and applied for the identification of functional SNPs associated with prostate cancer 67 . Two functional cell‐type‐specific SNPs and six functional cell‐type‐specific SNPs were identified in PC‐3 and LNCaP cells, respectively 67 .…”

“…The DiR‐seq was first developed and applied for the identification of functional SNPs associated with prostate cancer 67 . Two functional cell‐type‐specific SNPs and six functional cell‐type‐specific SNPs were identified in PC‐3 and LNCaP cells, respectively 67 . The same team not only identified prostate cancer risk‐associated SNPs in prostate cancer cell lines, 15,67 and dissected the regulatory mechanism of the risk SNP, but also performed applications for screening and evaluating functional SNPs associated with breast cancer 14 or autophagy 13 .…”

“…Two functional cell‐type‐specific SNPs and six functional cell‐type‐specific SNPs were identified in PC‐3 and LNCaP cells, respectively 67 . The same team not only identified prostate cancer risk‐associated SNPs in prostate cancer cell lines, 15,67 and dissected the regulatory mechanism of the risk SNP, but also performed applications for screening and evaluating functional SNPs associated with breast cancer 14 or autophagy 13 . Additionally, the application of the DiR‐seq system does not stop there, but these are enough to illustrate the accuracy and accessibility of the DiR‐seq system.…”