Coordinated change of a ratio of methylated H3-Iysine 4 or acetylated H3 to acetylated H4 and DNA methylation is associated with tissue-specific gene expression in cloned pig

Various cell types in higher multicellular organisms are genetically homogenous, but are functionally and morphologically heterogeneous due to the differential expression of genes during development, which appears to be controlled by epigenetic mechanisms. However, the exact molecular mechanisms that govern the tissue-specific gene expression are poorly understood. Here, we show that dynamic changes in histone modifications and DNA methylation in the upstream coding region of a gene containing the transcription initiation site determine the tissue-specific gene expression pattern. The tissue-specific expression of the transgene correlated with DNA demethylation at specific CpG sites as well as significant changes in histone modifications from a low ratio of methylated H3- lysine 4 or acetylated H3-lysine 9, 14 to acetylated H4 to higher ratios. Based on the programmed status of transgene silenced in cloned mammalian ear-derived fibroblasts, the transgene could be reprogrammed by change of histone modification and DNA methylation by inhibiting both histone deacetylase and DNA methylation, resulting in high expression of the transgene. These findings indicate that dynamic change of histone modification and DNA methylation is potentially important in the establishment and maintenance of tissue-specific gene expression.

Induction of Cytotoxic T Lymphocytes by Dendritic Cells Pulsed with Murine Leukemic Cell RNA / 대한혈액학회지

BACKGROUND: This study was done to assess the feasibility of dendritic cell generation from murine bone marrow and the efficacy of dendritic cells pulsed with total RNA to induce specific cytotoxic T lymphocyte response against leukemic cells. METHODS: Nucleated cells of inbred BALB/c mice were obtained and cultured with granulocyte/macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide (LPS) to induce dendritic cells. Total RNA of WEHI-3BD+, a myelomonocytic leukemia cell line from BALB/c, was transfected into the dendritic cells using liposome. RNA pulsed dendritic cells were irradiated and administered to the BALB/c mice intraperitoneally and splenic T lymphocytes were harvested. After restimulation with leukemic cells, T cell proliferation and specific cytotoxicity was assessed. RESULTS: Cells cultured with GM-CSF and lipopolysaccaride were found to have prominent dendritic processes. The percentage of cells showing high expression of both MHC class II and CD80, CD86, or CD11c was 69.6 %, 63.7%, and 41.8%, respectively. T cells stimulated by WEHI-3BD+ total RNA pulsed dendritic cells using DOTAP showed enhanced proliferation than those stimulated by total RNA or media only (P=0.05). When T cells were cocultured with WEHI-3BD+ as target cells, T cells stimulated by WEHI-3BD+ total RNA pulsed dendritic cells using DOTAP showed much increased cytotoxicity than controls. CONCLUSION: Dendritic cells pulsed with total leukemic RNA could stimulate T cells to induce specific cytotoxic effect.