Mechanism Investigation Of The Transcriptional Regulation Of Sp1 On Human NDRG2

Specificity protein 1 was first identified and cloned in 1983 by Dynan WS. Sp1 was shown to be a sequence-specific DNA-binding protein that activated abroad and diverse spectrum of mammalian and viral genes. Sp1 protein recognizes GC/GT boxes and interacts with DNA through three C2H2-type zinc fingers located at the C-terminal domain.As the initially reported gene in NDRG family, NDRG1(N-Myc down- regulated gene 1)augmented abnormally in the embryo of N-Myc knockout mouse. Therefore, NDRG1 was named as N-Myc downstream- regulated gene 1.Human NDRG2 was firstly cloned and reported by our lab (GenBank Number: AF 159092). Human NDRG2 locates at chromosome 14q11.2 and encodes a protein about 41KD which is composed of 357 amino acids. Since human NDRG2 shows about 60% homology to NDRG1,NDRG2 was also named as a N-myc downstream regulated gene 2.By bioinformatic analysis, We found there are two MZF-1(myeloid zinc finger 1, MZF-1) binding sites and four Sp1 binding sites. To investigate the transcriptional mechanism of MZF-1 and Sp1 on human NDRG2. pGL3-NDRG2(-1455——+274)and MZF-1 or Sp1 overexpression vector were transfected into HEK293 and Hela cells respectively. The cell samples were collected after 48hs and analyzed for dual-reporter gene assay. The results demonstrated that Sp1 enhanced the promoter activity of human NDRG2 3-4 folds. However, MZF-1 did not enhance the promoter activity of human NDRG2. Then, We constructed pSilencer-Sp1.By RT-PCR and Western blot, the result showed pSilencer-Sp1 reduced the expression of Sp1. At last pGL3-NDRG2(-1455——+274)and PEBGN-Sp1 or pSilencer-Sp1 were transfected into HEK293 and Hela cells respectively. The results demonstrated that PEBGN-Sp1 and pSilencer-Sp1 could repress the promoter activity of human NDRG2.To investigate the binding sites of Sp1 and NDRG2 core promoter, a series of different trunctions of NDRG2 and Sp1 overexpression vector were transfected into HEK293 cells. The results showed there were three Sp1 binding sites in the promoter of A:NDRG2(-148——-138),B:NDRG2(+15——+19),C:NDRG2(+29——+33). Then we constructed a series of mutant NDRG2 promoter (M1(A mutation),M2(B mutation),M3(C mutation),M12(AB mutation),M13(AC mutation)和M23(BC mutation)) by PCR. By dual-reporter gene assay,M1,M2 and M3 could partially repress the promoter activity of human NDRG2 compared to the control. While M12, M13, M23 could repress the promoter activity of human NDRG2 at least 50%. From the above results, we can't know how Sp1 regulates the promoter of NDRG2. To investigate whether Sp1 could interact with the core promoter of NDRG2 directly, We performed ChIP and EMSA, By ChIP assays,Sp1 could be recruited to the core promoter of NDRG2(-148——-138).From all the above results,we conclude that NDRG2 is a new target gene of Sp1. So our study not only improved the Sp1 signal pathway but also provide some meaningful clue and verification for the therapy of cancer.