PRECISE ALIGNMENT OF SITES REQUIRED FOR MU-ENHANCER ACTIVATION IN B-CELLS

The lymphocyte-specific immunoglobulin mu heavy-chain gene intronic en hancer is regulated by multiple nuclear factors. The previously define d minimal enhancer containing the mu A, mu E3, and mu B sites is trans activated by a combination of the ETS-domain proteins PU.1 and Ets-1 i n nonlymphoid cells. The core GGAAs of the mu A and mu B sites are sep arated by 30 nucleotides, suggesting that ETS proteins bind to these s ites from these same side of the DNA helix. We tested the necessity fo r appropriate spatial alignment of these elements by using mutated enh ancers with altered spacings. A 4- or 10-bp insertion between mu E3 an d mu(B) inactivated the mu enhancer in S194 plasma cells but did not a ffect in vitro binding of Ets-1, PU.1, or the mu E3-binding protein TF E3, alone or in pairwise combinations. Circular permutation and phasin g analyses demonstrated that PU.1 binding but not TFE3 or Ets-1 bends Ec enhancer DNA toward the major groove. We propose that the requireme nt for precise spacing of the mu A and mu B elements is due in part to a directed DNA bend induced by PU.1.