Precise alignment of sites required for mu enhancer activation in B cells.

The lymphocyte-specific immunoglobulin mu heavy-chain gene intronic enhancer is regulated by multiple nuclear factors. The previously defined minimal enhancer containing the muA, muE3, and muB sites is transactivated by a combination of the ETS-domain proteins PU.1 and Ets-1 in nonlymphoid cells. The core GGAAs of the muA and muB sites are separated by 30 nucleotides, suggesting that ETS proteins bind to these sites from these same side of the DNA helix. We tested the necessity for appropriate spatial alignment of these elements by using mutated enhancers with altered spacings. A 4- or 10-bp insertion between muE3 and muB inactivated the mu enhancer in S194 plasma cells but did not affect in vitro binding of Ets-1, PU.1, or the muE3-binding protein TFE3, alone or in pairwise combinations. Circular permutation and phasing analyses demonstrated that PU.1 binding but not TFE3 or Ets-1 bends mu enhancer DNA toward the major groove. We propose that the requirement for precise spacing of the muA and muB elements is due in part to a directed DNA bend induced by PU.1.     

One of two Ets-binding sites in the cytokeratin EndoA enhancer is essential for enhancer activity and binds to Ets-2 related proteins.

Expression of the mouse cytokeratin EndoA gene is restricted in endodermal and epithelial cells, and is regulated by an enhancer that is located 1 kilobase (kb) 3' downstream from the gene. The enhancer consists of six direct repeats, of which each contains two predicted Ets binding sites (EBS1 and EBS2) containing GGAA as a core. Mutation analysis showed that EBS1 is essential for the enhancer activity and additional effects of EBS2, suggesting that some Ets-related proteins bind and activate the enhancer through EBS1. We also showed that Ets-2 mRNA is expressed in PYS-2 cells and that Ets-2 protein produced by E. coli interacts with EBS1 but not with EBS2. Using co-transfection assays, we showed that Ets-2 can trans-activate the enhancer in PYS-2 cells. Mutations that impair Ets-2 binding abolished the activity of the EndoA enhancer. The results obtained from the binding competition assay using an Ets-2 specific antibody, however, suggest that EBS1 binds to an Ets protein which is distinct from Ets-2. These data show that Ets-2 related protein binds and activates the EndoA enhancer in a sequence-specific fashion.