Global Regulator SATB1 Recruits β-Catenin and Regulates TH2 Differentiation in Wnt-Dependent Manner
Figure 4
Competition between TCF and SATB1 for recruitment of β-catenin affects the TCF mediated transcription regulation.
(A) SATB1 does not interact with TCF proteins. Immunoprecipitation was performed as described in Materials and Methods. Nuclear extracts derived from human thymocytes treated with LiCl treated for 24 h were immunoprecipitated using anti-TCF-1 (lane 2) and anti-SATB1 (lane 3) followed by immunoblot with anti-SATB1. Similarly, immunoprecipitation was performed by incubating anti-TCF-4 with nuclear extracts from HEK 293T cells cultured in the presence (lane 6) and absence (lane 5) of LiCl for 24 h followed by immunoblot with anti-SATB1. Input (lanes 1 and 5) represents 5% of the nuclear extract used for IP. (B) In vitro competition assay was performed as described in Materials and Methods. TCF-4 contains all known functional domains of the TCF family proteins. Briefly, TCF-4 from nuclear extracts of Flag-TCF-4 transfected HEK 293 T cells was separately bound to GST-β-catenin (lanes 1–5) or GST-Arm (lanes 6–7) immobilized on glutathione-Sepharose. The bound complex was then incubated with various recombinant proteins as indicated, and the TCF-4 remaining bound to the column was monitored by Western blot analysis. “-” indicates that no protein was added and TCF-4 was eluted directly from the column. The upper panels represent WB analysis using anti-TCF-4, whereas the lower panel depicts Coomassie brilliant blue-stained SDS-polyacrylamide gel (15%) profile of various recombinant proteins used. (C) SATB1 and TCF-4 compete in vivo for association with β-catenin. Co-immunoprecipitation was performed to pulldown the SATB1:β-catenin complex from LiCl treated extracts from HEK 293T cells transfected with Flag-TCF4 (lane 2) or with empty vector (lane 1). IP and WB were performed using anti-SATB1 and anti-β-catenin, respectively. Expression of Flag-TCF4 and SATB1 was monitored by immunoblot analysis using respective antibodies as indicated in middle and lower panels. (D) SATB1 represses TCF targets in vivo. HEK 293T cells were transfected with indicated constructs for overexpression of full-length β-catenin (T41A), N-term β-catenin (1–567 aa), and SATB1 or empty vector in indicated combinations. β-catenin proteins encoded by both these constructs can translocate into nucleus and are therefore capable of transactivating Wnt/TCF/β-catenin targets; however, the N-term (1–576) protein does not interact with SATB1. RNA was isolated 48 h after transfection, and quantitative transcript profiling of TCF responsive genes integrin β1 and cyclin D1 was performed by real-time RT-PCR analysis as described in Materials and Methods.