PU.1 recruits a second nuclear factor to a site important for immunoglobulin kappa 3' enhancer activity.
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 kappa 3' enhancer (kappa E3'). Binding of PU.1 to the kappa E3' enhancer assists the binding of a second tissue-restricted factor, NF-EM5, to an adjacent site. Binding of NF-EM5 to kappa E3' 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 kappa E3' 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).
Pongubala
J M
JM
Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia 19104-6048.
Nagulapalli
S
S
Klemsz
M J
MJ
McKercher
S R
SR
Maki
R A
RA
Atchison
M L
ML
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004247:DNA; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004742:Enhancer Elements, Genetic; D007145:Immunoglobulin kappa-Chains / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism*; D009928:Organ Specificity / Q000235:genetics; D015682:Retroviridae Proteins, Oncogenic; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D007145:Immunoglobulin kappa-Chains; D009687:Nuclear Proteins; D015682:Retroviridae Proteins, Oncogenic; D014157:Transcription Factors; C062022:v-Spi-1 protein, Friend spleen focus-forming virus; D004247:DNA