PU.1 recruits a second nuclear factor to a site important for immunoglobulin κ 3' enhancer activity

Abstract

PU.1 is a B-cell- and macrophage-specific transcription factor. By an electrophoretic mobility shift assay and dimethyl sulfate methylation interference assays, we show that PU.1 binds to DNA sequences within the immunoglobulin κ 3' enhancer (κE3'). Binding of PU.1 to the κE3' enhancer assists the binding of a second tissue-restricted factor, NF-EM5, to an adjacent site. Binding of NF-EM5 to κE3' DNA sequences requires protein- protein interaction with PU.1 as well as specific protein-DNA interactions. This is the first known instance of PU.1 interacting with another cellular protein. NF-EM5 does not cofractionate with PU.1, suggesting that it is a distinct protein and is not a posttranslational modification of PU.1. UV- crosslinking studies and elution from sodium dodecyl sulfate-polyacrylamide gels indicate that NF-EM5 is a protein of approximately 46 kDa. Site-directed mutagenesis studies of the PU.1- and EM5-binding sites indicate that these sites play important roles in κE3' enhancer activity. By using a series of PU.1 deletion constructs, we have identified a region in PU.1 that is necessary for interaction with NF-EM5. This segment encompasses a 43-amino- acid region with PEST sequence homology, i.e., one that is rich in proline (P), glutamic acid (E), serine (S), and threonine (T).