Identification of an Sp1-like element within the immunoglobulin kappa 3' enhancer necessary for maximal enhancer activity

A number of functional DNA sequences have been identified within the murine immunoglobulin kappa 3' enhancer (kappa E3'). These DNA sequences were identified using plasmid reporter constructs in which the centrally active core region (or mutants of that region) of the enhancer was placed directly adjacent to the promoter of a reporter construct. Functional DNA sequences thus identified were found to bind to the transcription factors PU.1, NF-EM5, E2A, ATF-1, or CREM. In the studies reported here, we show that additional enhancer sequences that lie outside of the core region are necessary for maximal enhancer activity when the core region is not directly adjacent to the promoter. A series of progressive and internal deletion constructs shows that enhancer sequences between nucleotides 275 and 329 are important for enhancer activity, Progressive deletion to nucleotide position 329 resulted in a 4-fold reduction in enhancer activity. Using electrophoretic mobility shift assays, we shaw that this segment of the enhancer binds to ubiquitously expressed nuclear factors. Dimethyl sulfate methylation interference assays indicated protein-DNA interactions within a G-rich sequence between positions 302 and 306 and an A-rich sequence between positions 319 and 329. Ultraviolet light protein-DNA cross-linking studies revealed nuclear factors of approximately 85 and 105 kDa that bind to the newly identified enhancer region. Oligonucleotide competition studies and binding studies with purified Sp1 or Sp1 antibodies indicate that Sp1 can bind to this sequence. These studies show that functional sequences within the kappa E3' enhancer include an Sp1-like site approximately 90 bp 5' of the central 132 bp region originally believed to account for most of the enhancer activity.