Differentiation of stem cells from a pluripotent to a committed state involves global changes in genome expression patterns, critically determined by chromatin structure and interactions of chromatin-binding proteins. The dynamics of chromatin structure are tightly regulated by multiple epigenetic mechanisms such as histone modifications and the incorporation of histone variants. In the current work, we induced neural differentiation of a human embryonal carcinoma stem cell line, NTERA2/NT2, by retinoic acid (RA) treatment, primarily according to two different methods of adherent cell culture (rosette formation) and suspension cell culture (EB formation) conditions, and histone modifications and variations were compared through these processes. Western blot analysis of histone extracts showed significant changes in the acetylation and methylation patterns of histone H3, and expression level of the histone variant H2A.Z, after RA treatment in both protocols. Using chromatin immunoprecipitation (ChIP) coupled with real-time PCR, it was shown that these epigenetic changes occurred on the regulatory regions of 4 marker genes (Oct4, Nanog, Nestin, and Pax6) in a culture condition dependent manner. This report demonstrates the dynamic interplay of histone modification and variation in regulating the gene expression profile, during stem cell differentiation and under different culture conditions.
- Neural differentiation
- Retinoic acid