TAL1 does not homodimerize but forms heterodimers with class A bHLH proteins to bind DNA (17,18). seen in undifferentiating cells. These observations are consistent with the idea that TAL1 and Id1 have opposing effects on erythroid differentiation and that the level of TAL1/E2A heterodimer and/or another E protein-containing complex SPTAN1 may influence the decision of a cell to terminally differentiate. gene, which encodes the major tissue-specific bHLH protein expressed in the erythroid lineage (4,15), increases during Me2SO-induced differentiation of MEL cells (1,49). Overexpression of TAL1 Aciclovir (Acyclovir) enhances the spontaneous differentiation of MEL cells, whereas expression of antisense TAL1 RNA or a TAL1 mutant protein lacking the DNA binding basic region blocks chemically induced differentiation (1). Conversely, Idl messenger RNA is rapidly down-regulated in MEL cells during treatment with Me2SO (6,26) and forced expression of Idl blocks their differentiation (26,43). Although TAL1 and Idl do not heterodimerize with each other (17,45), each may form heterodimers with E proteins such as those encoded by the gene, E12 and E47/ E2-5 (5,17,50). By analogy with MyoD and Idl (21), TAL1 and Idl may compete for interactions with E proteins in erythroid cells such Aciclovir (Acyclovir) that the relative levels of active (TAL1/E) and inactive (Idl/E) complexes influence the expression of E-box-containing target genes involved in the switch to the differentiated state. No target genes for TAL1 have yet been identified in erythroid cells. A requirement for TAL1 in embryonic blood development in vivo has been established by targeted disruption of the mouse gene (38,40,42). Analysis of Aciclovir (Acyclovir) these mutants indicates that, in addition to its role during terminal erythroid differentiation, TAL1 also functions very early in hematopoiesis. TAL1 does not homodimerize but forms heterodimers with class A bHLH proteins to bind DNA (17,18). Surprisingly, genetic ablation of the mouse gene, which encodes potential dimerization partners of TAL1 in erythroid cells, has no apparent effect on erythropoiesis (52,53). This result suggests either that E2A proteins do not function in erythroid development and differentiation or that they are redundant with other E proteins in erythroid cells. Consistent with this notion of overlapping functions is the absence of any apparent defects in the erythroid lineage in Aciclovir (Acyclovir) mice carrying homozygous-targeted mutations in either the or gene (54). The E proteins HEB (20) and E2-2 (16) could potentially substitute for E2A to form dimers with TAL1. To elucidate the molecular mechanism(s) by which bHLH proteins regulate erythroid differentiation, we have examined the expression of E-box binding activities during Me2SO-induced differentiation of MEL cells. We present evidence for three activities in MEL cell nuclear extracts that increase in level by 12C24 h of induction. Using antisera against TAL1 and E-type bHLH proteins, we show that these inducible DNA binding activities contain E2A and TAL1, HEB, or an as yet unidentified protein. All three complexes are disrupted in vitro by addition of exogenous Idl and they are the only E-box binding activities so affected. Finally, in a cell line that constitutively overexpresses Id1, TAL1/E2A binding activity is definitely reduced. Surprisingly, no heterodimers of TAL1 and HEB or E2-2 were recognized in MEL cells either before or during differentiation. Together, these results suggest that the balance of heterodimers between E2A and TAL1 or additional bHLH proteins and the bad regulator Idl control terminal differentiation in the erythroid lineage. MATERIALS AND METHODS Cell Tradition, Nuclear Extract Preparation, and Electrophoretic Mobility Shift Assay MEL cells (subline DS19/sc9) and K562 cells (subline RA6) were managed and induced as explained previously (26). Stably transformed MEL lines C6 and B5 (26) were maintained in medium comprising 0.2 mg/ml G418. Nuclear components were prepared from ethnicities (0.25C1.0 1) using the method of Dignam et al. (11) with the help of protease inhibitors (2 for 20 min at 37C before addition of additional binding reaction parts. RESULTS MEL Cell Nuclear Components Contain Several E-Box Binding Activities That Differ in Their Affinities for Distinct E-Box Sequences MEL cells are known to transcribe several HLH genes (5,20,39,45,49), but translation of HLH proteins and their relationships in this system have been less well characterized. We while others have previously demonstrated that overexpression of the dominant-negative HLH Aciclovir (Acyclovir) protein Id1 blocks MEL cell differentiation in response to Me2SO (26,43), presumably by disrupting or preventing the formation of one or more DNA.