Proteomic Identification of TAL1/SCL-Interacting Proteins: ETO-2 and MTGR1 Interact with TAL1 in Erythroid Progenitors.

Abstract

The TAL1/SCL gene, originally identified from its involvement by a recurrent chromosomal translocation in T-cel acute lymphoblastic leukemia, encodes a basic helix-loop-helix (bHLH) transcription factor essential for hematopoietic and vascular development. Although TAL1 is believed to regulate transcription of specific sets of target genes, the mechanisms underlying TAL1-directed gene expression are poorly understood. Previous studies have shown, in fact, that it can act as either an activator or repressor depending on the coregulator(s) with which it interacts. To comprehensively identify TAL1's interaction partners in erythroid cells, we stably expressed a tandem epitope-tagged mouse TAL1 protein in murine erythroleukemia (MEL) cells and determined the composition of affinity-purified TAL1-containing complexes by multidimensional mass spectrometry. From this analysis, we identified all known members of a TAL1-containing DNA binding complex previously characterized in erythroid cells, including TAL1, its E protein DNA-binding partners, the zinc finger transcription factor GATA-1, the LIM-only protein LMO2, and the LIM domain-binding protein Ldb1, as well as proteins described to interact with GATA-1 (FOG-1), LMO2 (ELF2A2), and Ldb1 (SSDP2 and SSDP3). In addition, we identified a number of other DNA binding proteins, chromatin modifying proteins, and transcriptional regulators, including the ETO family members ETO-2 and MTGR1. TAL1 interaction with ETO-2 and MTGR1 was verified by coimmunoprecipitation analysis in MEL cells expressing these proteins at endogenous levels, in MEL cells stably expressing an epitope-tagged TAL1 protein, and in COS cells transiently transfected with TAL1 and ETO-2 or MTGR1 expression vectors. Mapping analysis with GAL4 fusion proteins identified the bHLH domain as the region in TAL1 responsible for interaction with these ETO family proteins. Significantly, expression of MTGR1 enhanced ETO-2 interaction with TAL1-GAL4 protein. Finally, transient transfection analysis with a luciferase reporter construct linked to multiple GAL4 DNA binding sites showed greater than additive augmentation of TAL1-directed gene repression with coexpression of the two ETO-related proteins compared to that observed with ETO-2 or MTGR1 transfected individually. These results identify ETO-2 and MTGR1 as authentic TAL1 interacting proteins and suggest that a hetero-oligomeric complex of the two contributes to TAL1-directed repression in erythroid progenitors.