Identification and characterization of stimulator of interferon gene silencer
10.3760/cma.j.cn112309-20201020-00483
- VernacularTitle:人干扰素基因刺激因子(STING)基因沉默子的克隆鉴定及功能初探
- Author:
Xiaoyu PANG
;
Haiyan CHEN
;
Linyuan CHEN
;
Huaguo XU
- From:
Chinese Journal of Microbiology and Immunology
2021;41(5):353-360
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To Clone the silencer sequences of human stimulator of interferon genes (STING) and evaluate its activity in HEK293T and HeLa, and to preliminarily investigate the transcriptional regulatory mechanisms, screening and verifying the possible binding elements for the silencer sequences of STING.Methods:The human STING-5-1a(-124~+ 267, transcription start site, TSS: 0) and STING-5-2a(-124~+ 168) regions were amplified by PCR, subcloned into pGL3-Basic plasmid, and the luciferase activity was detected in HEK293T and HeLa; the human STING silencer region STING-5-1b(+ 169~+ 267) was subcloned into the pGL3-Control plasmid (pGL3-C-5-1b-positive/negative) and STING-5-1b is divided into two complementary elements, subcloned into pGL3-Control vector, named pGL3-C-STING 5-1b-α(+ 169~+ 209) and pGL3-C-STING-5-1b-β(+ 210~+ 267), detecting the relative luciferase activity of the above plasmid in HEK293T and HeLa. Using bioinformatics methods to predict the transcription factor binding site of human STING silencer, make site-directed mutagenesis at the predicted binding site, and detect luciferase activity. Knockdown of THAP1 and TEAD1 by a siRNA strategy and detect the expression level of STING by western blot analysis. Chromosome immunoprecipitation assay further verified the binding of transcription factor to hSTING silencer.Results:It was verified that plasmids mentioned above were constructed correctly by nucleotide sequencing. The relative luciferase increased after truncating the STING-5-1b(+ 169~+ 267) fragment. The relative luciferase activity of pGL3-C-5-1b-positive recombinant plasmid decreased ( P<0.05), compared with pGL3-Control plasmid. Among them, STING-5-1b-β(+ 210~+ 267) fragment play a major inhibitory role. Using bioinformatics software to predict that the transcription factors PR domain zinc finger protein 4 (PRDM4), Thanatos-associated protein 1 (THAP1), TEA Domain Transcription Factor 1 (TEAD1), Nuclear receptor subfamily 4 group A member 1 (NR4A1), Krueppel-like factor 4 (KLF4) and Forkhead box protein O3(FOXO3) may bind to the human STING silencer region (+ 210~+ 267). After transfecting the mutant recombinant plasmid of the transcription factors into HEK293T and HeLa, the relative luciferase activity of THAP1-Mut and TEAD1-Mut were significantly increased, suggesting that STING silencers may contain binding sites of THAP1 and TEAD1. Knockdown of THAP1 and TEAD1 by a siRNA strategy significantly enhanced the transcription activity. Chromosome immunoprecipitation assay showed that the transcription factors TEAD1 and THAP1 combined with hSTING silencer region in the cells. Conclusions:The hSTING silencer luciferase reporter plasmid was successfully constructed. By the activity comparison, it is speculated that the core silencer region of human STING is located in the + 210~+ 267 element, which may contain several potential transcription factor binding sequences.The potential binding sites for transcription factors that may be contained in the DNA, and use Western blot and chromosome immunoprecipitation assays to further confirm the combination of transcription factors TEAD1 and THAP1 with hSTING silencer, laying the foundation for subsequent research.
