Figure 3

Chromatin structure of granulocyte-macrophage colony-stimulating factor (GM-CSF) transgenes. (A) Map of the human GM-CSF transgene, showing the positions of the GM-CSF gene and enhancer (E) and the locations of DNase hypersensitive sites (DHSs). (B) Analysis of DHSs in T lymphoblasts stimulated for 8 h with 20 ng/ml phorbol myristate acetate (PMA) and 2 μM A23187. Nil represents intact genomic DNA cleaved with Eco RI and the right hand lane in each panel is an Eco RI digest of DNA derived from DNase I-digested nuclei. The asterisks represent incomplete copies of the transgene. Mapping was performed using a Sal I-Eco RI fragment located within the GM-CSF gene. (C and D) Chromatin immunoprecipitation assays (ChIP) of tri-methylation of histone H3 K9 (C) and acetylation of histone H3 K9 (D) with non-specific immunoglobin G (IgG) as a ChIP antibody control. Assays were performed on cultured T cells previously activated with ConA, prepared from mouse lines A127 (open boxes) and J253 (closed boxes) after stimulation for 4 h with 20 ng/ml PMA and 2 μM A23187. Shown here are single representative experiments for lines A127 and J253 that have been replicated using lines M268 and D184, respectively.