Identification And Validation Of Transcription Regulated Genes Of Kinesin-like DNA Binding Protein

Background:Kinesin-like DNA binding protein (Kid) is a member of kinesin family (KIF). Kid regulates microtubule dynamics via its N-terminal of kinesin-like motor domain, and regulates transcription of its target genes via its C-terminal DNA-binding domain and nuclear localization signals (NLSs). As a microtubule motor protein, Kid is involved in spindle formation/function and chromosome movement/segregation during mitotic phases of cells. As a transcription factor, Kid might regulat expression level of ErbB-2.So far, the molecular structure of Kid and its functions based on the structure has been elucidated. However, the role of Kid in carcinogenesis and development of cancer was unknown. In the previous study of our group, the mRNA expression level of kinesin-like4(KLSL4), the encoding gene of Kid, was down-regulated in lymph node metastases compared to their paired primary breast cancers. Further study showed that the down-regulation of KNSL4mRNA was correlated to poor prognosis of breast cancer patients, indicating Kid may play an important role in carcinogenesis and development of breast cancer.Objective:To identify and validate kid target genes of transcriptional regulation, so as to gain insight into the role of Kid in carcinogenesis and development of breast cancer.Methods:DNA sequences occupied by Kid in MDA-MB-435S cells were profiled based on the chromatin immunoprecipitation combining with promoter microarray (ChIP-on chip). Using DNA selection and ligation (ChIP-DSL) method to prepare fluorescence-labeled targets of DNA enriched by anti-Kid antibody or non-antibody. The genes which were2-fold or more differentially enriched by anti-Kid antibody comparing with non-antibody were deemed as candidate genes. The candidate genes were validated by ChIP combining with real time PCR (ChIP-qPCR).Kid expression was knocked down in MCF-7cell by RNA interference targeting exon2and exon3respectively. Then MCF-7cell Subclones of Kid low-expression were identified by real time RT-PCR and western blot, and named as MCF-7/exon2-siRNA and MCF-7/exon3-siRNA. The expression levels of candidate genes in MCF-7/exon2-siRNA and MCF-7/exon3-siRNA cells were detected by real-time reverse transcription PCR (RT-PCR) assay.Results:1. Preparing targets using DSL method reduced initial ChIP-DNA to100ng. Compared with ligation mediate PCR (LM-PCR), DSL method is more sensitive and less costly to detect target genes of transcriptional factor.2. Two hundred and eighty seven candidate genes of Kid transcriptional regulated were obtained by using ChIP-on chip method. Of these genes,26were related to cell cycle,27to signal trasduction,40to transcription regulation,17to cell adhesion, and16to cell motility. Promoter DNA of ATF7IP was4.8-fold enriched by anti-Kid antibody than non-antibody, CDC25C was8.3-fold, PIP was11-fold, and TM4SF3was27.9-fold.3. ChIP-PCR assay showed that promoter of ATF7IP was4.9-fold more enriched respectively by anti-Kid antibody than non-antibody, CDC25C was10-fold, PIP was3-fold, and TM4SF3was11.8-fold.4. The mRNA expression level of Kid was decreased by80%and76%in MCF-7/exon3-siRNA and MCF-7/exon2-siRNA cells respectively compared with the parental cells. In consistent with mRNA expression level, the protein level of Kid was decreased by68%and52%respectively in MCF-7/exon3-siRNA and MCF-7/exon2-siRNA cells.5. The expression levels of ATF7IP and CDC25C mRNA in MCF-7/exon3-siRNA cells were3.5and10-fold up-regulated comparing with the parental cells. PIP and TM4SF3were not expressed in both MCF-7/exon3-siRNA and the parental cells.Conclusions:1. DSL is a sensitive method to preparing targets for promoter array hybridization.2. MCF-7subclones of Kid low-expression were established.3. Kid regulates transcriptions of ATF7IP and CDC25C genes negatively. Thus, it might be involved in carcinogenesis and development of breast cancer through transcriptional regulation of its target genes.