To characterize proteins that bind to the immunoglobulin (lg) heavy chain and the K light chain enhancers, an electrophoretic mobility shift assay with end-labeled DNA fragments was used. Three binding proteins have been found. One is NF-A, a factor found in all tested cell types that binds to the octamer sequence found upstream of all lg variable region gene segments and to the same octamer in the heavy chain enhancer. The second, also ubiquitous, protein binds to a sequence in both the heavy chain and the K enhancers that was previously shown to be protected from methylation in vivo. Other closely related sites do not compete for this binding, implying a restriction enzyme-like binding specificity. The third protein binds to a sequence in the K enhancer (and to an identical sequence in the SV40 enhancer) and is restricted in its occurrence to B cells. introduction lmmunoglobulin (lg) gene expression is governed by three types of tissue-specific regulatory sequences (Grosschedl and Baltimore, 1985)-the promoter (Falkner and Zachau, 1984; Bergman et al., 1984; Mason et al., 1985; Gopal et al., 1985; Foster et al., 1985; Picard and Schaffner, 1985), the enhancer (Gillies et al., 1983; Banerji et al., 1983; Neuberger, 1983; Mercola et al., 1983; Queen and Baltimore, 1983; Queen and Stafford, 1984; Picard and Schaffner, 1984), and, at least in the case of the p heavy chain gene, by an intragenic sequence as well (Grosschedl and Baltimore, 1985). Within the upstream promoter region, sequence comparisons (Parslow et al., 1984) followed by deletion analysis have indicated the importance of an octameric motif (ATTTGCAT), located at a characteristic distance upstream of all sequenced variable region genes (Falkner and Zachau, 1984; Bergman et al., 1984; Mason et al., 1985). We have recently reported the detection of a nuclear factor that interacts with this sequence (Singh et al., 1988). Enhancers were defined in viruses as regulatory sequences that can potentiate transcription from a variety of promoters in a distance-and orientation-independent manner, but the lg enhancers were the first found to be tissue-specific as well. It has been assumed that the action of these cis regulatory elements must be mediated by trans-acting factors, and there is now some experimental evidence to support this conjecture. Schdler and Gruss (1984) and Mercola et al.(1985) have carried out in vivo competition experiments which imply that the presumed factor can be functionally titrated by cotransfection of increasing amounts of enhancer sequences. Also, Ephrussi et al.(1985) and Church et al.(1985) have obtained footprints of a putative tissue-specific factor on the heavy chain enhancer in living cells and nuclei. Recently, a substantial advance has also been made by the development of enhancer-dependent in vitro transcription systems (Sassone-Corsi et al., 1984; Wildeman et al., 1984; Sergeant et al., 1984; Scholer and Gruss, 1985). Competition experiments carried out in vitro (Schliler and Gruse, 1985; Sassone-Corsi et al., 1985) have further indicated that enhancer function may be mediated by transacting factors. To understand the mechanisms of enhancer function and their role in the activation of tissue-specific genes, we have searched for the presence of such factors in nuclear extracts.

We report here interaction of factors with lg u and K enhancer sequence8 as detected by an electrophoretic mobility shift assay (Fried and Crothers, 1981; Garner and Revzin, 1981). This technique, based on the fact that nucleoprotein complexes are resolved from uncomplexed DNA by electrophoresis through polyacrylamide gels in low ionic strength buffers, has been elegantly …