The E2A-HLF oncoprotein activates Groucho-related genes and suppresses Runx1

The E2A-HLF fusion gene, formed by the t(17;19)(q22;p13) chromosomal transl ocation in leukemic pro-B cells, encodes a chimeric transcription factor co nsisting of the transactivation domain of E2A linked to the bZIP DNA-bindin g and protein dimerization domain of hepatic leukemia factor (HLF). This on coprotein blocks apoptosis induced by growth factor deprivation or irradiat ion, but the mechanism for this effect remains unclear. We therefore perfor med representational difference analysis (RDA) to identify downstream genet ic targets of E2A-HLF, using a murine FL5.12 pro-B cell line that had been stably transfected with E2A-HLF cDNA under the control of a zinc-regulated metallothionein promoter. Two RDA clones, designated RDA1 and RDA3, were di fferentially upregulated in E2A-HLF-positive cells after zinc induction. Th e corresponding cDNAs encoded two WD40 repeat-containing proteins, Grg2 and Grg6. Both are related to the Drosophila protein Groucho, a transcriptiona l corepressor that lacks DNA-binding activity on its own but can act in con cert with other proteins to regulate embryologic development of the fly. Ex pression of both Grg2 and Grg6 was upregulated 10- to 50-fold by E2A-HLF. I mmunoblot analysis detected increased amounts of two additional Groucho-rel ated proteins, Grg1 and Grg4, in cells expressing E2A-HLF. A mutant E2A-HLF protein with a disabled DNA-binding region also mediated pro-B cell surviv al and activated Groucho-related genes. Among the transcription factors kno wn to interact with Groucho-related protein, only RUNX1 was appreciably dow nregulated by E2A-HLF. Our results identify a highly conserved family of tr anscriptional corepressors that are activated by E2A-HLF, and they suggest that downregulation of RUNX1 may contribute to E2A-HLF-mediated leukemogene sis.