Epigenetic mark alterations on Oct4 regulatory region through embryonal carcinoma cell differentiation.

Favaedi, R.; Baharvand, H.; Shahhoseini, M.

Introduction: Embryonal carcinoma (EC) is a type of germ cell tumor that occurs in the ovaries and testes. The EC cells are the malignant counterparts of embryonic stem (ES) cells and provide a useful alternative to embryos for the study of mammalian cell differentiation. Transcription profile of both ES and EC cells undergoes remarkable changes onset of differentiation Materials and Methods: Pluripotency genes, expressed in undifferentiated state of maintained cells, are down regulated upon differentiation. One of these genes is POU domain homeobox gene of Oct4 which has a long up-stream regulatory region (2600 bp), consisting of proximal enhancer (PE), distal enhancer (DE) and proximal promoter (PP). This widespread regulatory region is target of many histone modifications as epigenetic marks which pronounced rapid changes by differentiation. The aim of this research was comparative evaluation of epigenetic profile of Oct4 regulatory region in embryonal carcinoma In order to this aim a human EC cell line termed NT2, originated from testis tumor, grown under two different adherent and non-adherent culture conditions. Then differentiation induced to cells by retinoic acid (RA) treatment for 3 days. Regulatory regions of Oct4 gene studied by chromatin immunoprecipitation (ChIP) method coupled with real-time PCR technique to compare histone modifications on these regions as the epigenetic marks after differentiation. Results: Results showed that by induced 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. Moreover comparison of the two culturing systems revealed 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 importance of epigenetic marks of gene regulatory regions especially proximal ones. Also, current findings showed the effective role of cell culture condition on the epigenetic regulation of gene expression.

EPIGENETICS; CELL differentiation; EMBRYONAL tumors; EMBRYONIC stem cells; TRANSCRIPTION factors; GENE expression

Iranian Journal of Reproductive Medicine, 2013, p48

1680-6433

Academic Journal