Development of a high efficient promoter finding method based on transient transfection

Yao Lu; Qilong Li; Kexin Zheng; Chenghao Fu; Chunying Jiang; Dayu Zhou; Chao Xia; Shiliang Ma

doi:10.1016/j.gene.2019.100008

Development of a high efficient promoter finding method based on transient transfection

Gene X. 2019 Feb 12:2:100008. doi: 10.1016/j.gene.2019.100008. eCollection 2019 Jun.

Authors

Yao Lu¹, Qilong Li¹, Kexin Zheng², Chenghao Fu¹, Chunying Jiang¹, Dayu Zhou², Chao Xia¹, Shiliang Ma¹

Affiliations

¹ College of Bioscience and Biotechnology, Shenyang Agricultural University, Shenyang, Liaoning, PR China.
² College of Food Science and Technology, Shenyang Agricultural University, Shenyang, Liaoning, PR China.

Abstract

In metazoan genome, the mechanism of gene expression regulation between transcriptional regulatory elements and their target gene is spatiotemporal. Active promoters possess many specific chromosomal features, such as hypersensitive to DNaseI and enrichment of specific histone modifications. In this article, we proposed a novel method which possesses a high efficiency to find promoters in vitro. A promoter-trap library was constructed with totally 706 random mouse genomic DNA fragment clones, and 260 promoter-active fragments of the library were screened by transient transfection into 4T1 cells. To demonstrate the accuracy of this promoter finding method, 13 fragments with promoter activities were randomly selected for published DNase-seq and ChIP-seq data analysis, downstream transcripts prediction and expression confirmation. qRT-PCR results showed that six predicted transcription units were successfully amplified in different mouse tissues/cells or in reconstituted mouse mammary tumors. Our results indicate that this promoter finding method can successfully detect the promoter-active fragments and their downstream transcripts.

Keywords: ATAC-seq, Assay for transposase-accessible chromatin using sequencing; Bioinformatics; CAGE, cap analysis of gene expression; CMV, Cytomegalovirus; Cancer-specific promoter; ChIP-seq, Chromatin immunoprecipitation followed by massively parallel DNA sequencing; Ct, threshold; DHS, DNaseI hypersensitive sites; DNase-seq, DNase I hypersensitive sites sequencing; EF1a1, eukaryotic translation elongation factor 1 alpha 1; FBS, fetal bovine serum; GRO-seq, global run-on sequencing; Gene expression regulation; Gene finding; H3K4me3, histone H3 lysine 4 trimethylation; Itpr2, inositol 1, 4, 5-triphosphate receptor 2; LSINCT5, long stress-induced non-coding transcript 5; MCS, multiple cloning site; MPRA, Massively parallel reporter assays; Mouse breast cancer; PBS, phosphate buffered solution; Promoter trap; RNA-seq, RNA sequencing; SD, standard deviation; STARR-seq, Self-transcribing active regulatory region sequencing; TFs, transcription factors; TSS, transcription start sites; dNTPs, deoxy-ribonucleoside triphosphate; eRNAs, enhancer RNAs; mSEAP, mouse synthetic secreted embryonic alkaline phosphatase; pNPP, p-nitropheny-phosate; qRT-PCR, quantitative RT-PCR; tpk1, thiamine pyrophosphokinase.