The ETS Protein ELF2 Contributes to a Novel TAL1/SCL-Containing DNA-Binding Complex That Positively Regulates Erythroid Gene Expression and Cell Differentiation,

Abstract 3389

The TAL1/SCL gene, originally discovered from its involvement by a recurrent chromosomal translocation in T-cell acute lymphoblastic leukemia, is important for hematopoietic stem cell and erythroid, megakaryocyte, and macrophage progenitor function and is essential for hematopoietic and vascular development. Although Tal1 can bind DNA with one of its E protein DNA binding partners, its function in erythroid cells has been thought to be mediated by a larger complex recognizing an E box-GATA or GATA sequence motif. In independent lines of investigation, a novel DNA-binding motif for TAL1 comprised of an ETS site immediately adjacent to an E box was identified by site selection analysis in K562 cells, and multiple peptides derived from a single ETS protein, E74-like factor 2 (Elf2, also known as NERF), were identified by tandem mass spectrometry analysis of Tal1-containing protein complexes purified from murine erythroleukemia (MEL) cells. To determine whether Elf2, previously shown to interact with Tal1's interaction partner LMO2, recognized this sequence element, electrophoretic mobility shift analysis was carried out using MEL cell nuclear extracts. This revealed the existence of a novel multiprotein ETS-E box DNA-binding complex containing Tal1, Elf2, Lmo2, the LIM domain-binding protein Ldb1, and Ldb1-interacting single-stranded DNA binding proteins Ssbp2 and Ssbp3. Structure-function analysis of this DNA-binding activity, which increased with chemical induction of MEL cell differentiation, showed that both the E box and ETS site were required for ternary complex formation, that preference existed for specific ETS flanking sequences, and that precise spacing between the two transcription factor binding sites was critical for complex formation. Finally, a physiological target for this complex was identified in the Ssbp3 gene, with Tal1, Elf2, and Ldb1 found to specifically co-occupy the third intron of this gene by chromatin immunoprecipitation analysis but not a similar ETS-E box element in the 11th intron. In accord, Ssbp3 expression, ETS-E box DNA-binding activity, and hemoglobin concentration increased with enforced expression of Elf2 in MEL cells, while Ssbp3 protein abundance, ETS-E box DNA-binding activity, and cellular differentiation decreased with Elf2 knockdown. These studies identify a novel TAL1- and ELF2-containing DNA-binding complex in erythroid progenitors that positively regulates gene expression and differentiation. In addition, they reveal a previously unknown role for the ETS protein ELF2 in erythropoiesis.