Real-time PCR assays (Figure 1) measure steady-state levels of ɛy mRNA, not transcriptional activity of the ɛy promoter. To investigate whether Sox6 directly acts on the ɛy gene promoter at the transcriptional level, we used an in vitro transient transfection assay and GM979 cells, a murine erythroleukemic cell line that expresses both ɛy and adult beta globins [30]. We generated an ɛy promoter reporter construct (E-Luc) by fusing a micro-LCR (μLCR) element (2.5 kb) [31] to the ɛy proximal promoter (2.2 kb), followed by the luciferase reporter gene, as shown in Figure 2A (detailed in Materials and Methods). Overexpression of Sox6 in GM979 cells by transient transfection leads to a dosage-dependent repression of E-Luc reporter activity (Figure 2B). In contrast, overexpression of a truncated Sox6 protein that lacks its HMG domain [32] (similar to the p 100H mouse allele) fails to repress E-Luc activity (Figure 2B). These data indicate that Sox6 acts to repress the ɛy promoter at the transcriptional level.