ATF-2 and CREB augment endogenous γ-globin transcription in K562 and primary erythroid cells. (A) Site-directed mutagenesis was completed in the G-CRE at base -1225 (G→A) (m1) and bases -1226 and -1227 (AC→TG) to produce the m2 mutant reporter. Steady-state ^Gγ-promoter activity was decreased for m1 and abolished in the m2 reporter compared with wild type (wt). The star above the graph (comparison between the wt and mutant reporters) represents significance at the P < .05 level. (B) The ability of NaB (2 mM) and TSA (0.5 μM) to activate γ-promoter transcription in the mutant reporters was tested. The reporters were transfected into K562 cells, induced with NaB or TSA for 24 hours, and then luciferase activity was measured. (C) The ability of CREB and ATF-2 to trans-activate the mutant ^Gγ-promoters was analyzed. Both proteins were able to trans-activate the m1 reporter, whereas the m2 mutation abolished this effect. (D) CREB and ATF-2 activate endogenous γ-globin gene transcription. Studies were completed to determine endogenous γ-globin transcription after enforced protein expression (see “Materials and methods”). The cells were harvested 24 and 48 hours later, and qPCR analysis was completed to quantify γ-globin mRNAlevels as a ratio to the internal control GAPD. Data are shown as the fold increase in γ-globin/GAPD expression relative to untreated sample (mean ± SEM). (E) Enforced CREB and ATF-2 expression activates γ-globin in primary erythroid progenitors. Peripheral blood mononuclear cells were grown in a two-phase liquid culture system. Progenitor cells were transfected on day 4 during phase 2 using a Nucleofector device transfection system (see “Materials and methods”). The level of γ-globin, β-globin, and GAPD gene expression was quantified by qPCR. The ratio of γ/β globin mRNA was calculated after the level of mRNA for each gene was divided by GAPD mRNA; the ratios were normalized to values obtained for untransfected erythroid progenitors.