FIG. 2.
The BTB domain is physically and functionally critical for interactions with corepressors. (A) PLZF contains discrete functional domains, including the N-terminal BTB domain, a less-well-characterized second repression domain (RD2), and nine C-terminal Zn fingers (ZF). The PLZFΔBTB construct used in these experiments is also shown. (B) Coimmunoprecipitations (IP) performed in 293T cells. Cells (4 × 105) were transfected with 1 μg of PLZF or PLZFΔBTB expression vectors. Endogenous N-CoR was precipitated, and the resulting fraction was immunoblotted to detect PLZF. Expression of both proteins is confirmed by direct Western blotting of the cell lysates as shown in the left two lanes. The lower band in the PLZF lanes is an alternate PLZF product commonly seen in cells transfected with the PLZF expression vector. (C) Coimmunoprecipitations with endogenous SMRT similar to panel B. (D) 293T cells (2 × 105) were transfected with 200 ng of pCDNA, pCDNA-PLZF, or pCDNA-PLZFΔBTB, with or without 150 ng of CMX-SMRT. The cells were cotransfected with 50 ng of a luciferase reporter containing four PLZF binding sites from the IL-3Rα chain promoter and a tk-renilla internal control. Results are expressed as fold repression of luciferase normalized to the internal control.