P-88: Comparing Epigenetic Profile of Oct4 Regulatory Region in Embryonal Carcinoma Cells under Retinoic Acid Induction

Background: Embryonal carcinoma (EC) cells derived from germ cell tumors are valuable tools for investigating differentiation and developmental biology processes in vitro. The advantage of the reproducible and rapid expansion of these cell lines provides a useful alternative to embryos for the study of mammalian cell differentiation. During early stages of cell differentiation, the rate of transcription of large numbers of genes is substantially altered in a time-dependent manner. Oct4 is a POU domain homeobox gene, expressed in undifferentiated embryonal carcinoma and embryonic stem cells and is quickly down regulated upon induction of differentiation. Transcriptional repression of Oct4 is followed by pronounced epigenetic changes on the regulatory region of the gene. Oct4 has a long up-stream regulatory region of about 2600 bp, consisting of proximal enhancer (PE), distal enhancer (DE) and proximal promoter (PP). Materials and Methods: In this study we induced differentiation of a human embryonic carcinoma cell line, NT2, under two different adherent and non-adherent culture conditions. Using chromatin immunoprecipitation coupled with real-time PCR technique we compared histone modifications as the epigenetic marks on the regulatory region of Oct4 gene after 3 days of differentiation. Results: The data shown that after induction of differentiation the repressive epigenetic marks of hypoacetylation and methylation on lysine-9 of histone H3 occurred very effectively on the up-stream of Oct4, especially in PP region. Also, comparing the two culturing systems it was shown that methylation of lysine-9 of H3 histone was more drastic in PE region of adherent cells rather than suspension cells. This epigenetic profile was in agreement with the difference observed in the expression level of Oct4 in these two culturing systems. Conclusion: The current study clearly shows the effective role of cell culture condition on the epigenetic regulation of gene expression.