EMSA and ChIP Assays Show that Sox6 Directly Binds to the epsilony Promoter
Sox6 might repress the epsilony promoter, either through direct physical contact with the promoter or by regulating intermediates affecting the epsilony promoter. To investigate whether Sox6 is directly associated with the epsilony promoter, we first performed electrophoretic mobility shift assays (EMSA) using a c-Myc-tagged Sox6 in a reticulocyte lysate-based transcription/translation in vitro system. The probes used are listed in Figure 3A. The 36-base pair (bp) WT probe corresponds to the critical region of the epsilony promoter defined in our promoter deletion analyses. This probe contains two consensus Sox/Sox6 binding sites. Also included in our EMSA are three mutated probes that are, either truncated (M1), or mutated (M2 and M3) in Sox/Sox6 binding sites (Figure 3A). Sox6 is able to physically associate with the 36-bp region (Figure 3B) within the epsilony promoter defined by the deletion analysis experiments (Figure 2C). The 36-bp probe was shifted by the tagged Sox6 protein. Moreover, both c-Myc and Sox6 antibodies supershift the band, indicating that the binding is Sox6-specific. To rule out the possibility that the c-Myc tag itself binds to the probe, an HA-tagged Sox6 was used in another EMSA that confirmed these results (Figure 3C). 
Next, nuclear extracts from MEL cells were used in EMSA employing the same 36-bp probe. MEL cells, a murine erythroleukemic cell line, express adult beta globins, but not epsilony [33]. Sox6 directly binds to this DNA sequence in MEL cells (Figure 3D). The intact consensus Sox/Sox6 binding sites of the DNA probe are required for the binding, as shown in the competition assay (Figure 3E). Ablation of putative Sox/Sox6 binding sites (M1 and M3) abolish their ability to compete in EMSA (Figure 3E). The M2 mutant probe may compete partially with WT binding. 
To investigate the functional significance of the intact Sox/Sox6 binding sites, the epsilony promoter reporter constructs with mutagenized Sox/Sox6 binding sites were co-transfected with the Sox6 overexpression vector into GM979 cells. Consistent with the EMSA results, the mutant epsilony promoter reporter constructs (with either one or both Sox/Sox6 binding sites mutagenized) do not result in significant promoter repression in transfection studies (Figure 3F). Thus, both sites are required for maximal repression of epsilony by Sox6, but not to the same degree. 
We also tested whether Sox6 binds to the epsilony promoter in vivo using chromatin immunoprecipitation (ChIP) (Figure 4). The Sox6-containing complex was immunoprecipitated from MEL cells or from liver cells of 15.5 dpc WT mice using Sox6 antibody. Figure 4 shows that the epsilony proximal promoter is readily immunoprecipitated with Sox6 antibody in both MEL cells and liver cells. Normal IgG was used as a negative control (Figure 4A). The above data (Figures 3 and 4) clearly indicate that Sox6 acts as a repressor of the epsilony gene by directly binding to the epsilony promoter, probably as a dimer. 
