Preliminary Research About The Mechanism Of Nuclear Import Of Ccctc-binding Factor

CTCF is a ubiquitously expressed and evolutionarily conserved zinc finger (ZF) protein that binds target sites through combinatorial use of its 11 ZFs in eukaryote, which plays diverse roles including inhibition and activation of gene expression, gene imprinting and insulator function. Researchers concentrated their attention to the function of CTCF, but its nuclear translocation has never been researched. As an important regulation factor interrelated to cell proliferation and tumorgenesis, CTCF nuclear translocation is an important process.CTCF, which is a high molecular weight protein, needs its nuclear location signal (NLS) to recognize its transit receptor and then is transported into nucleus. Nearly all transport of proteins appears to be mediated by any member of the karyopherin family. The purpose of our research is to find a certain member of the karyopherin family which interacts with CTCF. Moreover, we preliminarily study the nuclear import mechanism and regulation mode of CTCF. We also study the relationship between nuclear import of CTCF and the invasive capability of tumor cells.Our study was based upon the data of gene chips. We looked for the target genes of CTCF by ChIP-on-chip. The results showed that the promoter region of importin13 (ipo13 or imp13) is the target site of CTCF. Yusufzai and Felsenfeld et al purified CTCF and its co-factors by co-IP. The Importinα3 (karyopherinα4 or kpna4) was identified by mass spectrometry in Purified complexes. According to all about, our study first indicated the relationship between CTCF distribution and the invasive capability of tumor cells by immunofluorescence. The results showed that CTCF mostly located in nucleus in low invasive tumor cells and located in cytoplasm in highly invasive tumor cells.Then we further confirmed that CTCF bind at the promoter region of ipo13 by ChIP-PCR. We detected the mRNA and protein level of CTCF in HeLa and HeLa-CTCF-II-11, which is a CTCF knock-down cell line, by RT semi-quantitative PCR and western blot. The results showed that the expression of IPO13 and KPNA4 decreased in HeLa-CTCF-II-11 cells, which indicated that CTCF positively regulate IPO13 and KPNA4 genes expression.Also, we used the methods of co-IP and GST PULL-DOWN to detect the interactions between CTCF and IPO13 and between CTCF and KPNA4. In GST PULL-DOWN, we constructed recombinant plasmid p GEX-4T-1-IPO13 and p GEX-4T-1- KPNA4 and expressed recombinant proteins in E coli. CTCF from 293FT cells lysate and wheat germ in vitro expression system interacted with GST-IPO13 and GST-KPNA4. Co-IP experiment also indicated that there is an interaction between IPO13 and CTCF.Taken together, we found CTCF self-regulate its nuclear import process through the expression regulation of IPO13 and KPNA4 genes.