FIGURE 3.

SATB2 occupies ^G/Aγ-globin promoters in K562 cells. A, schematic representation of the human β-globin locus indicating the locations of ChIP primers (see also supplemental Fig. S3) and 3C fragments (HindIII fragments) used in this study (see Fig. 5, E and F). B, ChIP analysis of myc-SATB2 binding status at the γ-globin gene promoter in K562 cells ectopically expressing Myc-tagged SATB2 using an anti-Myc antibody. Serial dilution was employed for semi-quantitative assay. C, ChIP analysis using anti-SATB2 antibody (Ab51502) shows native SATB2 binding at the γ-globin gene promoter in K562 cells. (**, p < 0.01). D, restriction enzyme assay to show proportion of ^G/Aγ-globin promoters in the SATB2 ChIP samples before and after immunoprecipitation. The result indicates relative enrichment of the ^Gγ promoter after immunoprecipitation. The PCR product amplified from the ^Gγ promoter was digested by MseI into 225-bp and 44-bp bands, whereas that of the ^Aγ promoter formed an uncut 265-bp band in the assay. Bottom: MAR- ^G/Aγ-pro sequence across the MseI site. E and F, EMSA analysis of in vitro SATB2 binding at MAR-^G/Aγ-pro. The biotinylated DNA probes derived from MAR-^G/Aγ-pro were incubated with purified GST-SATB2 in the absence or presence of unlabeled competitors, with GST as a negative control. E, competitors were serially mutated MAR-^Gγ-pro with underlined bases changed to CCGGCC or (F) wild-type and mutated MAR-^A/Gγ-pro corresponding to mut6 of MAR-^Gγ-pro. The arrows indicate the band shift position of the SATB2-MAR complex. G, luciferase reporter analysis of the SATB2 regulatory effect on transcriptional activities of wild-type and mutated ^G/Aγ- promoters in 293T cells. Each error bar represents a standard deviation calculated from experiments performed in triplicate. Student's t test was used for statistical analysis, and p values are indicated (**, p < 0.01).