text: Transfection Studies Using GM979 Cells Indicate That Sox6 Directly Represses the epsilony Gene Promoter at the Transcriptional Level
  Real-time PCR assays (Figure 1) measure steady-state levels of epsilony mRNA, not transcriptional activity of the epsilony promoter. To investigate whether Sox6 directly acts on the epsilony 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 epsilony and adult beta globins [30]. We generated an epsilony promoter reporter construct (E-Luc) by fusing a micro-LCR (muLCR) element (2.5 kb) [31] to the epsilony 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 p100H mouse allele) fails to repress E-Luc activity (Figure 2B). These data indicate that Sox6 acts to repress the epsilony promoter at the transcriptional level. 
  Sox6 has been shown to act as a repressor and to interact with a widely expressed co-repressor, CtBP2, on the fgf-3 promoter [5]. CtBP2 is expressed in GM979 cells (unpublished data). To investigate whether the interaction with CtBP2 is required for Sox6 repression of the epsilony promoter, we introduced a point mutation (L386H) in the Sox6 protein that has been previously reported to be sufficient to abolish Sox6-CtBP2 interaction [5]. This amino acid change is not in the HMG DNA binding domain. However, this mutant version of Sox6 retains the ability to repress the epsilony promoter in the transfection assay (Figure 2B), indicating that Sox6 represses the epsilony promoter in a CtBP2-independent manner. Deletion analysis of the epsilony promoter, as shown in Figure 2C, defined a region (-63 to -37) within the epsilony proximal promoter that is critical for Sox6 repression. Analysis of this short region reveals two Sox/Sox6 consensus binding sites [5] (Figure 3A). 
  