| Restin was a novel gene isolated by our lab from differentiation tumor cell induced by all-trans retinoic acid in 1999, and its molecular functions were not well understood. It mainly expresses in terminally differentiated cells. By transfection of Restin into tumor cell, the cells were arrested in G1 phase and the cell proliferation was inhibited. It implies that restin may be correlated to cell cycle regulation. Chomez and colleague also found this gene by homologous sequence splicing in the database of gene bank in 2001 and named as MAGE-HI, but there are no any biological functions of Restin reported.Our previous research identified that the Restin was a 219 amino acid residue alkaline protein, and contained a bipartite nuclear targeting sequence and a MHD {MAGE homology domain, MHD) located in the N-terminal and C-terminal region respectively. About the MHD of Restin, it is homologous to MAGEs within 30-49%, such as Necdin and MAGE-D, the homolog is 49% and 38% respectively. And more, the restin is localized on the X chromatosome in Xp11.22 site, same as most MAGE family gene. So, Restin seems to be a novel member ofMAGE superfamily, however, the nucleic acid sequence of restin displays no homologous to any MAGEs. The phylogenetic analysis of MAGE family proteins and Restin shows that the relationship of Restin is nearer to type II, such as MAGE-D1 and Necdin, rather than type I MAGE members. The tissue expression analysis of Restin shows that Restin same as Necdin and MAGE-D, is high expressed in prostate, testis, nervous tissue and fetal tissues, and low or less in the white blood cells and tumor tissues.The tumor cells transfected with restin are arrested in Gl phase and the cell proliferation is inhibited. It implies that restin may be correlated to cell cycle regulation. Other data showed that the Necdin, a homolog of Restin, could suppress cell growth by interacting with transcription factor E2F1 and p53. Morever, Tcherpakov and colleague found that the MAGE-D1, Necdin and MAGE-HI (Restin) could interacted with p75. So, we deduced that the Restin most likely regulated cell cycle by interacting with some transcription factors. Gl phase is the only stage that cells can receipt the signal of proliferation and growth inhibition and many molecules could produce a marked effect in the stage. A lot of transcription factors such as ATF, c-fos, c-jun, E2F and p53 were envolved in this stage.In this study, we try to identify the transcription factors interacted with Restin by using mammalian cell two-hybrid system.At first, total 31 transcription factors correlated to cell proliferation, differentiation, physiological stress and apoptosis, were selected (see in article) to use in the two-hybrid, and cloned into plasmid pACT by using RT-PCR to amplify these transcription factors from fetal tissue cDNA or stimulated lymphocyte cDNA.As result, only six transcription factors ATF3, ATF4, E2F6, C-Jun, C-Fos and p53 were correctly cloned.And then, all six pACT-TFs had been detected whether they could produce autoluminescence and could interact with restin by cotransfecting with pBIND or pBlND-restin into COS-7 cell respectively. The results showed that all six TFs displayed noautoluminescence in this two-hybrid system, and one of them, the ATF3 displayed a strong interaction with restin.To verify the interaction of Restin and ATF3, a series of truncated fragments of ATF3 were inserted into pACT to be used in the further study. Four truncated fragments of ATF3 have been cloned and hybridized with restin in two-hybrid system: ATF3-AD (contains transcriptional activation domain), ATF3-BD (contains DNA binding domain), A ATF3(contains complete transcriptional activation domain and part of binding domain) and A ATF3-BD(contains part of binding domain near C-terminal). The results showed that ATF3-BD could interact with Restin, but ATF3-AD did not. And A ATF3-BD reduced the activity of interaction with Restin. It suggested that Restin most likely interacted with ATF3 binding domai...
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