Identification and Functional Study of G-quadruplexes in <i>SENP1</i> Promoter

doi:10.13523/j.cb.1905003

China Biotechnology

2020, Vol. 40

Issue (1-2): 92-101 DOI: 10.13523/j.cb.1905003

Orginal Article

Identification and Functional Study of G-quadruplexes in SENP1 Promoter

ZHOU Yan-xing,HAN Meng,LIU Na-nv,HUANG Wei-wei(

)

College of Life Science, Northwest A & F University, Yangling 712100, China

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Abstract

Objective: The regulation effect of G-quadruplexes (G4) on the SUMO-specific proteases 1 (SENP1) transcriptional expression was studied. Method: Reporting plasmids of SENP1 promoter were constructed by cloning different SENP1 promoter fragments. The core transcriptional regulatory region of SENP1 promoter was identified by using reporter assay. The sequence of core transcriptional regulatory region of SENP1 promoter was analyzed and G-quadruplex (G4) formation sequence was predicted. Oligonucleotides of G4 formation sequence from SENP1 promoter were synthesized, and their topological structure was detected by circular dichroism analysis. G4 ligand TMPyP4 and G4 helicase G4R1 were used to detect the regulatory effect of promoter G4 on SENP1 transcriptional expression by using reporter assay and Western blot. Result: The -910~+226 region was found to be the core transcriptional regulatory region of SENP1 promoter. Sequence analysis showed the core region of SENP1 promoter is rich in G/C and contains G4 formation sequences. Circular dichroism analysis confirmed that the oligonucleotides of G4 formation sequence from SENP1 promoter form G4 structure. Reporter assay and Western blot showed that promoter G4 inhibited the transcriptional expression of SENP1. Conclusion: G4 exist in the core transcriptional regulatory region of SENP1 promoter and show negative regulation on SENP1 transcriptional expression, which provides new research ideas and experimental clues for revealing the mechanism of SENP1 in physiological and pathological processes.

Key words： SENP1 G-quadruplexe TMPyP4 G4R1

Received: 05 May 2019 Published: 27 March 2020

ZTFLH:

Q291

Corresponding Authors: Wei-wei HUANG E-mail: whuang0210@163.com

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	Yan-xing ZHOU
	Meng HAN
	Na-nv LIU
	Wei-wei HUANG

Cite this article:

ZHOU Yan-xing,HAN Meng,LIU Na-nv,HUANG Wei-wei. Identification and Functional Study of G-quadruplexes in SENP1 Promoter. China Biotechnology, 2020, 40(1-2): 92-101.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.1905003 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I1-2/92

Table 1 PCR primers sequences

Fig.1 SENP1 promoter reporter plasmids and reporter assay (a) Schematic representation of the SENP1 promoter and the primers used for SENP1 promoter clone (b) The diagram of SENP1 promoter reporter constructs and related Gluc activity

Fig.2 The sequence analysis and quadruplex forming sequences prediction of SENP1 promoter (a) The G/C content of 1 000bp region upstream transcription start site (TSS) of SENP1 promoter and partial its 5'-UTR The TSS is designated as +1 (b) The quadruplex forming sequences prediction of SENP1 promoter (c) The sequence of -200~+60 region of SENP1 promoter and predicted quadruplex forming sequences (underlined)

Fig.3 Oligonucleotides of G4 forming Sequences from SENP1 promoter and circular dichroism (CD) analyses (a) The oligonucleotides of G4 forming sequences from SENP1 promoter and their corresponding mutant (M) The G-tracts with G4 structure forming potential are underlined, G in middle of G-tracts were replaced by A in mutant oligonucleotides SP-1M and SP-2M individually. pu27 was used as a positive control, which is a G4 forming sequence in the c-Myc promoter and has been characterized to form G4 structure (b) Circular dichroism spectroscopy analyses of oligonucleotides derived from the SENP1 promoter The absorption spectra of different oligonucleotides were analyzed by circular dichroism chromatography after annealing in TE solution containing K⁺ at 95℃. The abscissa represents the wavelength of polarized light and the ordinate represents the absorptivity. The vertical line represents the absorption peak

Fig.4 Effects of G4 ligand TMPyP4 on the SENP1 expression (a) Reporter assay to detect the effect of TMPyP4 on SENP1 transcription The SENP1 promoter reporter plasmid was transfected into Hep-2 cells. The transfected cells were treated with different dose of TMPyP4 for 48 hours,and then the Gluc activity of the culture medium was detected (b) Western blot to detect the effect of TMPyP4 on SENP1 protein level Hep-2 cells were treated with different dose of TMPyP4 for 48 hours. Protein samples were collected for Western blot and beta-actin for control

Fig.5 The effect of G4R1 on the SENP1 expression (a) Reporter assay to detect the effect of G4R1 on SENP1 transcriptional expression The SENP1 promoter reporter plasmid, Flag-G4R1 expression plasmid with different DNA quantities,and SEAP expression plasmid were co-transformed into Hep-2 cells for 48 hours, and the Gluc and SEAP activity of the culture medium were detected (b) Western blot to detect the effect of G4R1 on SENP1 protein level Flag-G4R1 expression plasmids with different DNA quantities were transfected into Hep-2 cells, and protein samples were collected for Western blot and beta-actin for control


[1]	Eifler K, Vertegaal A C O . SUMOylation-mediated regulation of cell cycle progression and cancer. Trends Biochem Sci, 2015,40(12):779-793.

[2]	Yang Y, He Y, Wang X , et al. Protein SUMOylation modification and its associations with disease. Open Biol, 2017,7(10):170167.

[3]	Mukhopadhyay D, Dasso M . Modification in reverse: the SUMO proteases. Trends Biochem Sci, 2007,32(6):286-295.

[4]	Cheng J, Kang X, Zhang S , et al. SUMO-specific protease 1 is essential for stabilization of HIF1α during hypoxia. Cell, 2007,131(3):584-595.

[5]	Yamaguchi T, Sharma P, Athanasiou M , et al. Mutation of SENP1/SuPr-2 reveals an essential role for desumoylation in mouse development. Mol Cell Biol, 2005,25(12):5171-5182.

[6]	Yu L, Ji W, Zhang H , et al. SENP1-mediated GATA1 deSUMOylation is critical for definitive erythropoiesis. J Exp Med, 2010,207(6):1183-1195.

[7]	Xia N, Cai J, Wang F , et al. SENP1 is a crucial regulator for cell senescence through deSUMOylation of Bmi1. Sci Rep, 2016,6:34099.

[8]	Xu Y, Zuo Y, Zhang H , et al. Induction of SENP1 in endothelial cells contributes to hypoxia-driven VEGF expression and angiogenesis. J Biol Chem, 2010,285(47):36682-36688.

[9]	Cai R, Yu T, Huang C , et al. SUMO-specific protease 1 regulates mitochondrial biogenesis through PGC-1α. J Biol Chem, 2012,287(53):44464-44470.

[10]	Wang Q, Xia N, Li T , et al., SUMO-specific protease 1promotes prostate cancer progression and metastasis. Oncogene, 2013,32(19):2493-2498.

[11]	Zhang W, Sun H, Shi X , et al. SENP1 regulates hepatocyte growth factor-induced migration and epithelial-mesenchymal transition of hepatocellular carcinom. Tumour Biol, 2016,37(6):7741-7748.

[12]	Mu J, Zuo Y, Yang W , et al. Over-expression of small ubiquitin-like modifier proteases 1 predicts chemo-sensitivity and poor survival in non-small cell lung cancer. Chin Med J (Engl), 2014,127(23):4060-4065.

[13]	Wang X, Liang X, Liang H , et al. SENP1/HIF-1α feedback loop modulates hypoxia-induced cell proliferation, invasion, and EMT in human osteosarcoma cells. J Cell Biochem, 2018,119(2):1819-1826.

[14]	Wang Z, Jin J, Zhang J , et al. Depletion of SENP1 suppresses the proliferation and invasion of triple-negative breast cancer cells. Oncol Rep, 2016,36(4):2071-2078.

[15]	Dong B, Gao Y, Kang X , et al. SENP1 promotes proliferation of clear cell renal cell carcinoma through activation of glycolysis. Oncotarget, 2016,7(49):80435-80449.

[16]	Bawa-Khalfe T, Cheng J, Wang Z , et al. Induction of the SUMO-specific protease 1 transcription by the androgen receptor in prostate cancer cells. J Biol Chem, 2007,282(52):37341-37349.

[17]	Wang C, Tao W, Ni S , et al. Tumor-suppressive microRNA-145 induces growth arrest by targeting SENP1 in human prostate cancer cells. Cancer Sci, 2015,106(4):375-382.

[18]	Chen S Y, Teng S C, Cheng T H , et al. miR-1236 regulates hypoxia-induced epithelial-mesenchymal transition and cell migration/invasion through repressing SENP1 and HDAC3. Cancer Lett, 2016,378(1):59-67.

[19]	Zhou G Q, Han F, Shi Z L , et al. miR-133a-3p targets SUMO-specific protease 1 to inhibit cell proliferation and cell cycle progress in colorectal cancer. Oncol Res, 2018,26(5):795-800.

[20]	Rhodes D, Lipps H J . G-quadruplexes and their regulatory roles in biology. Nucleic Acids Res, 2015,43(18):8627-8637.

[21]	Huppert J L, Balasubramanian S . Prevalence of quadruplexes in the human genome. Nucleic Acids Res, 2005,33(9):2908-2916.

[22]	Kwok C K, Merrick C J . G-Quadruplexes: prediction, characterization, and biological application. Trends Biotechnol, 2017,35(10):997-1013.

[23]	Schaffitzel C, Berger I, Postberg J , et al. In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei. Proc Natl Acad Sci USA, 2001,98:8572-8577.

[24]	Maizels N, Gray L T . The G4 genome. PLoS Genet, 2013,9(4):e1003468.

[25]	Siddiqui-Jain A, Grand C L, Bearss D J , et al. Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-MYC transcription. Proc Natl Acad Sci USA, 2002,99(18):11593-11598.

[26]	Dai J, Dexheimer T S, Chen D , et al. An intramolecular G-quadruplex structure with mixed parallel/antiparallel G-strands formed in the human BCL-2 promoter region in solution. J Am Chem Soc, 2006,128(4):1096-1098.

[27]	Cogoi S, Xodo L E . G-quadruplex formation within the promoter of the KRAS proto-oncogene and its effect on transcription. Nucleic Acids Res, 2006,34(9):2536-2549.

[28]	Neidle S, Parkinson G . Telomere maintenance as a target for anticancer drug discovery. Nat Rev Drug Discov, 2002,1(5):383-393.

[29]	Bugaut A, Balasubramanian S . 5'-UTR RNA G-quadruplexes: translation regulation and targeting. Nucleic Acids Res, 2012,40(11):4727-4741.

[30]	Lee J Y, Yoon J, Kihm H W , et al. Structural diversity and extreme stability of unimolecular Oxytricha nova telomeric G-quadruplex. Biochemistry, 2008,47(11):3389-3396.

[31]	Asamitsu S, Obata S, Yu Z , et al. Recent progress of targeted G-Quadruplex-preferred ligands toward cancer therapy. Molecules, 2019,24(3):E429.

[32]	Mendoza O, Bourdoncle A, Boule J B , et al. G-quadruplexes and helicases. Nucleic Acids Res, 2016,44(5):1989-2006.

[33]	Chen MC, Murat P, Abecassis K , et al. Insights into the mechanism of a G-quadruplex-unwinding DEAH-box helicase. Nucleic Acids Res, 2015,43(4):2223-2231.

[34]	Paramasivan S, Rujan I, Bolton P H . Circular dichroism of quadruplex DNAs: applications to structure, cation effects and ligand binding. Methods, 2007,43(2):324-331.

[35]	Huang W, Smaldino P J, Zhang Q , et al. Yin Yang 1 contains G-quadruplex structures in its promoter and 5'-UTR and its expression is modulated by G4 resolvase 1. Nucleic Acids Res, 2012,40(3):1033-1049.

[36]	Renciuk D, Rynes J, Kejnovska I , et al. G-quadruplex formation in the Oct4 promoter positively regulates Oct4 expression. Biochim. Biophys. Acta, 2017,1860(2):175-183.

[37]	Grand C L, Han H, Munoz R M , et al. The cationic porphyrin TMPyP4 down-regulates c-MYC and human telomerase reverse transcriptase expression and inhibits tumor growth in vivo. Mol Cancer Ther, 2002,1(8):565-573.

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