Fig. 4

Direct involvement of STAT6 in targeting DNA demethylation in specific dendritic cell (DC) genes. a Chromatin immunoprecipitation assays confirm STAT6 binding to two genes (DUOX1, SLAMF1) that become specifically demethylated in DC differentiation. The panel also shows the loss of STAT6 to DC-specific genes following treatment with JAK3 inhibitor PF-956980. Lack of binding of STAT6 under the conditions of macrophage (MAC) differentiation (also in the presence of PF-956980) is also shown. b Diagram showing STAT6 gene domains and the double mutant that mimics phosphorylated STAT6 (STAT6VT) and leads to a gain of function. c Western blot of 293 T cells transfected with wild-type STAT6 (STAT6 WT) and the activating double mutant STAT6VT showing the presence of the protein in the cytosolic or nuclear fraction. d Fluorescence-activated cell sorting analysis showing the green fluorescent protein (GFP) labelling of cells infected with a GFP-expressing lentiviral MIG vector (pCDH-MIG) containing STAT6VT (right panel) and empty GFP-expressing MIG vector as a negative control (left panel). e CD209 mean fluorescence measured by flow cytometry in monocytes (MO) infected with STAT6 VT and the MOCK control vector after 9 days of culture with granulocyte macrophage colony-stimulating factor (GM-CSF). Analysis was done by setting a gate to select cells with high GFP expression. f Effects on the DNA methylation levels of two DC-specific genes in MOs treated with GM-CSF for 48 h followed by infection with mock or STAT STAT6V. Both the GFP+ and GFP− fractions are shown. Examples of two DC-specific genes (left), MAC-specific genes (middle) and two genes demethylated in both DC and MAC differentiation (right) are shown. g Model depicting the participation of the IL-4-JAK3-STAT6 pathway in targeting demethylation of DC-specific genes