Figure 7. Gsc is a transcriptional target of Mixl1.

(A) Schematic diagram of the Gsc promoter region for transient transfection assays (−831 to +123). Boxes 1 and 2 represent the two pvariant MBSs, gMBS1 (−453 to −443) and gMBS2 (−370 to −360), respectively. gMBS1 is located within the conserved distal element (DE; from − 456 to −429), while gMBS2 is within the proximal element (PE; from −396 to −346). Below the diagram are the 26-bp EMSA probes (top strand sequences shown) that correspond to −461 to −436 (containing gMBS1 or its mutant, gMBS1M) and −378 to −353 (containing gMBS2 or its mutant, gMBS2M) regions of the Gsc gene, respectively. The TAAT/ATTA half sites are marked by inverted arrows and the mutations are underlined. (B) EMSA of [³²P]-labeled, double-stranded MBS, gMBS1 and gMBS2 probes with increasing amounts of Mixl1 HD (10 nM for lanes 2, 7, and 12; 50 nM for lanes 3–5, 8–10, and 13–15). Lanes 1, 6, and 11 contained no protein. Mixl1 HD and each of the probes formed two complexes (I and II). Binding to [³²P]-labeled MBS, gMBS1 and gMBS2 probes was efficiently competed by unlabeled MBS, gMBS1, and gMBS2 sequences, respectively (lanes 4, 9, and 14), but not by sequences containing MBS M3 (lane 5), gMBS1M (lane 10) or gMBS2M (lane 15). (C) pGL3-based luciferase reporter constructs for analyzing the promoter region (−831 to +123) of the Gsc gene (GscPro). Positions of gMBS1 and gMBS2 are marked by open boxes and mutant gMBS1 and gMBS2 marked by “X”. pGL3-GscProM1 and pGL3-GscProM2 contained mutations in gMBS1 and gMBS2, respectively, while pGL3-GscProM3 contained both mutations in both gMBS1 and gMBS2. (D) i-Mixl1 ES cells were transfected with pGL3-basic or the pGL3-GscPro based plasmid (GscPro, GscProM1, GscProM2, or GscProM3) and then cultured in LIF-free medium in the presence or absence of DOX (0.4 μg/ml). Induction of i-Mixl1 (+DOX) resulted in significant activation of the reporter luciferase gene in cells transfected with by pGL3-GscPro. Little or no activation of the reporter gene was detected in cells transfected by the other plasmids (pGL3-GscProM1, pGL3-GscProM2, and pGL3-GscProM3). (E) Specificity of the anti-Mixl1 antibody for the ChIP assay was validated by immunoprecipitation of FLAG-Mixl1 from COS cells transfected with an FLAG-Mixl1 expression plasmid (pMT23-FLAG-Mixl1). Cell lysates were incubated with the anti-Mixl1 antibody. Immunocomplexes were purified using protein A agarose and subjected to Western blotting with an anti-FLAG antibody. FLAG-Mixl1 was identified in cells transfected with pMT23-FLAG-Mixl1 (arrowhead) but not the empty pMT23 vector. IP, immunoprecipitation; Blot, Western blotting. (F and G) ChIP assays. i-Mixl1 ES cells were cultured in LIF-free medium in the presence or absence of DOX (0.4 μg/ml) for 1–3 days followed by in vivo crosslinking with formaldehyde. Cell lysates were sonicated and sheared chromatin was subjected to immunoprecipitation with a nonspecific rabbit IgG, anti-Mixl1 antibody (anti-Mixl1), or anti-acetyl-Histone H3 (anti-H3). (F) Quatitative real-time PCR analysis of immunoprecipitated DNA from DOX-induced (+DOX) and untreated (-DOX) differentiating (days 1–3) i-Mixl1 ES cells. The relative amount of immunoprecipitated DNA (relative IP) was determined using the 2^{−ΔΔ Ct} method. (G) Conventional PCR analysis of immunoprecipitated DNA from DOX-induced (+DOX) and untreated (−DOX) differentiating (day 2) i-Mixl1 ES cells. PCR products were resolved by 2.5% agarose gel electrophoresis. In, input (1%); IP, immunoprecipitated DNA. PCR-amplified promoters are marked by arrows and the antibody or IgG for immunoprecipitation is listed by each panel.