Abstract
The lymphocyte-specific immunoglobulin μ heavy-chain gene intronic enhancer is regulated by multiple nuclear factors. The previously defined minimal enhancer containing the μA, μE3, and μB sites is transactivated by a combination of the ETS-domain proteins PU.1 and Ets-1 in nonlymphoid cells. The core GGAAs of the μA and μB 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 μE3 and μB inactivated the μ enhancer in S194 plasma cells but did not affect in vitro binding of Ets-1, PU.1, or the μE3-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 μ enhancer DNA toward the major groove. We propose that the requirement for precise spacing of the μA and μB elements is due in part to a directed DNA bend induced by PU.1.