Identification Of The DNA Binding Modules Of SIPA1 Protein And Its Target Genes

SIPA1(signal-induced proliferation-associated protein 1)is a mitogen-induced GTPase activating protein.It has been reported that SIPA1 is involved in metastasis and occurrence of a variety of malignant cancers.More interestingly,SIPA1 protein has been found locating in the nucleus of the malignant breast cancer cells and interacting with DNA,activating the integrin ?1/CD44 promoter activity,leading to cancer cell metastasis.These findings suggested that the nuclear SIPA1 protein might act as a transcription factor.So,it is necessary to clarify that wheather SIPA1 protein could bind to DNA directely or not.What is the DNA motif that could bind to the SIPA protein? Which genes contain these motifs? Which domain on SIPA1 protein could bind to the motifs?In order to determine the DNA-bound domain on SIPA1 protein,the primary structure,secondary structure and the tertiary structure of the known regions of SIPA1 were firstly analyzed by bioinformatics method.The predicted results indicate that there is a coiled-coil(972 aa-1034 aa)domain on C-termine of SIPA1 protein.Many publications have been proved that the coiled-coil domain is one of the basic structures that can bind to DNA directly and regulate gene expression in the cell.It is highly posible that SIPA1 protein could bind to DNA directly.Then,to determine the direct binding of SIPA1 protein to DNA,an in vitro DNA-protein binding assay was employed.The Sipa1 truncated fragements were constructed into p GEX4T-1,which named as plasmids p GEX4T-1-? N(540-1042aa),p GEX4T-1-PDZ(540-763aa)and p GEX4T-1-CC(764-1042aa).The peptides of these trunctcated fragements were expressed and purified.By used of Electrophoretic Mobility Shift Assay(EMSA)assay,the C terminal ?N region of SIPA1 were proved to bind to DNA directly.Next,in order to find the DNA motif that SIPA1 protein binds to,the Systematic Evolution of Ligands by Exponential Enrichment(Selex)was used to identify the potential motifs.After multiple rounds of PCR amplification,the fragments with high affinity to SIPA1 protein were amplified and enriched from the synthezed DNA library.Fifty-nine DNA sequences binding to SIPA1 were obtained.Further more,by searching and aligning these sequences,the most conserved bases which could be the DNA motif bingding to SIPA1 protein were identified as GCACGAT/CG/ATNNNTG/A.Impotantely,106 promoter regions containing this motif were discoved in human promoter database,in which five of them were also the cadidates from the published SIAP1 Ch IP-sequencing data in metastatic breast cancer cells,indicating that this motif might be the site that mediates the binding of SIPA1 protein to these target genes.To demonstrate wheather the CC domain of SIPA1 protein binds to DNA fragments directly,the prufied GST-SIPA1 truncated peptides were used to detect the bingding of the promoter region of ITGB1 gene,which contains the motif,by EMSA.The results showed that DNA shifting was blocked by the CC domain,while no blocking signal was observed in other group of peptides,indicating that the CC domain on the SIPA1 C-terminal may mediate its directly binding to DNA.In summary,the interaction mode between SIPA1 protein and DNA was firstly clarified,and the target genes directly regulated by SIPA1 protein was further identified in this thesis.These findings are helpful to reveal the multiple roles of SIPA1 as a newly transcription factor to regulate cell adhesion,migration or metastasis in cancers.