Increased CREB-dependent promoter activity in AML blasts in vivo is associated with H3K9me3 levels at CRE-binding sites. (A) Primary CD34⁺ (n = 3) and AML blasts (n = 10) were transiently transfected with a CRE-driven reporter gene construct (or empty vector as a control; data not shown) and cultured for 6 hours before luciferase activity was determined. Transfection efficiency was normalized to renilla luciferase activity. To analyze the effect of stimulation of the cAMP pathway, forskolin was added after transfection and samples were analyzed accordingly (diagram on the right). In the absence (P < .01) and presence (P < .01) of forskolin, CRE-driven reporter activity was significantly higher in AML blasts. Indicated are mean ± SEM (Mann-Whitney U test). (B) PCA is shown that groups AML and CD34⁺ progenitor cells according to their H3K9me3 levels at genomic loci with CREs. Indicated are also AML specimens with a more “CD34-like” higher H3K9me3 level at CRE sites and those with “true AML-like” lower H3K9me3 levels at CRE sites. (C) Luciferase reporter assays were performed to identify differences in CRE-driven promoter activities within AML samples. AML samples were selected because of their H3K9me signature either close to the CD34⁺ pattern or to the AML pattern and analyzed for CRE-dependent promoter activity in transient transfection assays. The samples used for analysis are indicated in Figure 4B. “Control” depicts AML blasts (with AML-like H3K9me3 level) transfected with a promoter-less luciferase construct. AML patients with a CD34-like H3K9me3 pattern showed only minimal CREB-dependent promoter activity, whereas AML blasts with an “AML-like” H3K9me3 pattern showed more than 60-fold higher CREB-dependent promoter activity. Similar analyses carried out in the presence of forskolin to stimulate the cAMP-response pathway are indicated on the right. Indicated are mean ± SD.