FIGURE 5.

ASH2L promotes the enhancer activity of the ERα gene through GATA3. A, the diagram of ERα regulatory elements. The six promoters are indicated from A to F along with two enhancers (EN1 and EN2). Enhancer 1 (EN1) was used for luciferase activity and ChIP assays. B, depletion of ASH2L decreased the activity of ERα enhancer. ASH2L-depleted cells (sh2 as described above) or wild type cells expressing control shRNA (control) were transfected with control luciferase reporter (pGL-Luc) or the luciferase reporter directed by ERα enhancer (pGL-ER-Luc) and β-gal expression vector. Luciferase activity was normalized to β-gal activity. **, p < 0.01 (versus control reporter). ##, p < 0.01 (versus control shRNA). C, depletion of GATA3 decreased activity of the ERα enhancer. BT483 cells were infected with viruses expressing two different shRNAs targeting GATA3 (sh1, sh2) or control shRNA (NS). Real-time RT-PCR was performed to determine GATA3 mRNA levels (left panel). GATA3-depleted cells or wild type cells expressing control shRNA (control) were transfected and analyzed as described in B (right panel). ##, p < 0.01 (versus control shRNA); **, p < 0.01 (versus control reporter). D, ASH2L activated ERα enhancer through GATA3. Immortalized mammary epithelial cells were transfected with pGL-ER-Luc or mutant pGL-ER-Luc reporters, β-gal expression vector, plus ASH2L expression vector and GATA3 expression vector as indicated. ##, p < 0.01 (versus control); **, p < 0.01 (versus normal reporter).