Assessment Of Differentiation And Progression Of Benign And Malignant Hepatic Tumors Using Array Based Comparative Genomic Hybridization

152 Background The E2F transcription factor family is known to play a key role in the cell cycle progression and proliferation. But limited number of genes, which are transcriptionally regulated by this gene, has been identified. To clarify genome-wide screening of E2F1-binding DNA sequences at the promoter of putative E2F1-target genes in living human cells, we used a combination of chromatin immunoprecipitation (ChIP) and in-house human genome array (chip) composed of bacterial artificial chromosome clones (BACs). Materials and Methods In a manner of ChIP-on-chip, from a glioblastoma cell line, T98G, cross-linked protein-DNA complexes were precipitated with anti-E2F1 antibody, and total input DNA was obtained in the same manner without the antibody. Each of the DNAs was amplified by adaptor ligation-PCR and labeled with Cy3 and Cy5. The same volume of labeled DNA was competitively hybridized with BACs spotted on our custom-made BAC array. Results We detected many positive spots including BACs containing cyclinA2 and N-myc, which are well known as the transcriptional targets for E2F1. Among them we chose strongly positive BACs. These BACs contain known genes but not recognized as E2F1 targets, and these genes harbor E2F1 consensus recognition sequences within 2000 nucleotides spanning the transcription start site. Those three BACs contained 11 genes with E2F1 consensus sequences, and of them, only four genes were expressed in T98G cells. Conventional ChIP assay demonstrated in vivo binding of E2F1 to the consensus sequences of all four genes. One of them, ETG1 (lab. name) is known to be an apoptosis regulator. The transcriptional activity of the luciferase reporter constructs containing the consensus E2F1 binding site of ETG1 were elevated by co-transfection of E2F1. Exogeneously overexpressed E2F1 increased endogeneous ETG1 mRNA level. Conclusion The E2F1 pathway in a mechanism of ETG1 regulation may function within cancer cells to enhance tumor progression. Our “ChIP on chip” on BAC-array platform can facilitate to identify DNAs interacting with the specific proteins including transcription factors.