FIG. 10.

PEX1 is a GATA-4 cofactor. (A) Mapping of the DNA elements required for GATA-4/PEX1 synergy. HeLa cells were cotransfected with wild-type (Wt), mutated PERE (PERE mut), and mutated GATA (GATA mut) −695-bp ANF promoter luciferase constructs and PEX1, GATA-4, or both expression vectors. (B) GATA elements are sufficient to mediate GATA-4/PEX1 synergy. HeLa cells were cotransfected with a minimal BNP promoter driven by multimerized GATA binding sites (GATA3x) and increasing amount of GATA-4 in presence or absence of PEX1 expression vector. (C) PEX1 directly interacts with GATA-4 in vitro. Luciferase and GATA-4 were translated and ³⁵S labeled; LacZ, SRF, and PEX1 were produced in bacteria as MBP fusion, and the in vitro pull-down assays were performed as described in Materials and Methods. MBP-SRF and MBP-LacZ were used as positive and negative controls for GATA-4 interaction. (D) Mapping of GATA-4 domains required for PEX1 synergy. HeLa cells were cotransfected with the −695 ANF promoter luciferase reporter and different GATA-4 expression vectors: wild-type GATA-4 (1 to 440), C-terminal-deleted GATA-4 (ΔC, 1 to 332), N-terminal-deleted GATA-4 (ΔN, 210 to 440), and N- and C-terminal-deleted GATA-4 (ΔC/ΔN, 210 to 332) or with a point mutation in the second zinc finger (ZFm), with or without PEX1. All GATA-4 constructs were described previously (9). In panels A, B, and D, the data show the means ± the SEM of at least four independent determinations.