Functional Research Of Transcription Factor CTCF

CTCF is an exceptionally conserved transcription factor which is widely expressed in eukaryotics. CTCF interacts with multiple genes through the recognition of different DNA targets by variable combinational usage of 11 zinc fingers within M domain. CTCF is related to multiple functions, such as repression of c-myc gene promotor and activation of app gene promotor, silence of chicken lysozyme gene, functional reading of H19/Igf2 imprinting marks, interaction with insulator element of different gene domains, and participation in X-inactivation.It's the first study which aimed to repress CTCF expression in somatic cell lines by siRNA intereference and carry out the subsequent global analysis of CTCF-regulated gene candidates.In our study, CTCF cDNA was amplified from total RNA of Hela cells and was ligated into pGEM-T Easy plasmid. N domain and M domain of CTCF, termed CTCFN and CTCFM respectively, was successfully expressed in E.coli BL21(DE3) using pET expressing system. Then CTCFN was purified by Ni-NTA agarose column, the purity of CTCFN being more than 80%; we also did some research on in-column renaturation of CTCFM by gel filtration and on purification of CTCFM by denaturative Ni-NTA agarose column. Antibody to CTCFN was elicited by immunizing rabbit, then purified and used to detect CTCF level by Western blotting. The specificity of CTCF antibody can meet the requirement of this assay.Subsequently, we successfully decreased the expression of CTCF in two different mammalian cell lines by RNA interference. In-vitro transcription prepared siRNA repressed CTCF mRNA and protein level, and exhibited cell growth inhibition. The degree of repression depended on different experimental conditions. Moreover, endoribonuclease-prepared siRNA, esiRNA, exhibited dramatic repression of CTCF mRNA, protein and cell growth. It seems to be some correlation between repression of CTCF mRNA, protein and inhibition of cell growth.- 4 -We also demonstrated that the transcription activation activity of full length CTCF is less than that of domain N by in vitro transient transfection assay. So the transactivation activity of CTCF protein is mainly due to CTCFN in our assay.Our results has given important experimental evidence and set up essential technique for the global study of CTCF related expression profile after repression by siRNA interference in somatic cell lines.