The LIM domain-binding protein Ldb1 is known to form higher order complexes with LIM-homeodomain and LIM-only (LMO) proteins to regulate diverse developmental programs, including hematopoiesis. The level of Ldb1 is critical for its cellular roles, and its turnover is regulated by the E3 ubiquitin ligase RLIM. Single-stranded DNA-binding protein (SSDP), an Ldb1-interacting partner, is an essential gene for embryonic development and has been shown to regulate axis formation in Xenopus and wing development in Drosophila through Ldb1; however, the mechanisms by which SSDPs regulate these and other developmental programs are still obscure. We previously reported that a DNA-binding complex containing the basic helix-loop-helix protein TAL1/SCL, its DNA-binding partner E47, zinc finger protein GATA-1, LIM domain protein LMO2, and Ldb1 stimulates Protein 4.2 (P4.2) transcription in erythroid progenitors through tandem E box-GATA elements in the gene’s proximal promoter. We have now established that SSDP2 is associated with this complex (by supershift analysis) and occupies the promoter of this gene (by chromatin immunoprecipitation analysis) in murine erythroleukemia (MEL) cells. Further, enforced expression of SSDP2 in these cells stimulated P4.2 reporter activity and accumulation of P4.2 and beta-globin mRNAs, and cotransfection of SSDP2 with the five originally identified components of this complex further increased promoter activity in reporter analysis. Importantly, overexpression of SSDP2 in MEL cells significantly increased Ldb1 protein half-life and steady-state levels of Ldb1 and LMO2 protein. This effect on Ldb1 stability required the Ldb1-interacting domain of SSDP2, consisting of its first 94 amino acids (SSDP2(1–94)), and was also observed in Cos7L and CHO cells. We showed, in addition, that SSDP2 or SSDP2(1–94), but not an Ldb1 interaction-defective mutant, prevented RLIM-mediated degradation of both Ldb1 and LMO2 in transfected cells, that SSDP2 protection of LMO2 degradation required Ldb1, and that SSDP2 directly inhibited RLIM-mediated ubiquitination of Ldb1. Immunoprecipitation analysis revealed that overexpression of SSDP2 or SSDP2(1–94) significantly decreased interaction between Ldb1 and RLIM. Finally, SSDP2 protein expression in differentiating MEL cells paralleled this multi-protein DNA-binding activity and overexpression of SSDP2 in these cells dramatically increased E box-GATA DNA-binding activity, with maximal formation of the ternary complex requiring coexpression of SSDP2, Ldb1, and LMO2. Together, these studies reveal a positive role for SSDP2 in erythroid gene expression and identify a biochemical function for SSDP2 in regulating Ldb1 stability and transcriptional complex assembly. The mechanism mediating Ldb1 stabilization appears to involve competitive inhibition of RLIM interaction with Ldb1.

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