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).

to 20 min. Utricles were explanted to chilled Medium-199 containing 25 mM Hepes buffer and Hanks' salts (Gibco). The sensory epithelia were isolated, and the otolithic membranes were removed with fine forceps. The culture chambers contained small wells made from cover glasses and Polyallomer rings 9 mm in diameter attached with Silastic adhesive. They were coated with Cell-Tak (Collaborative Research) before one or two utricles were placed in each well with 50 gl of medium. The medium consisted of Medium-199 with Earle's salts, 26 mM sodium bicarbonate, 25 mM Hepes, 0.69 mM L-glutamine (Gibco), supplemented with 20% fetal bovine serum (FBS) (Gibco), penicillin (10 units/mI) and Fungizone (25 ng/mi). Cultures were maintained at 37rC in a 5% CO2 environment. Guinea pigs become sexually mature at 4 to 8 weeks of age [J. E. Wagner and P. J. Manning, The Biology of the Guinea Pig (Academic Press, New York, 1976), p. 9]. Seven of the specimens were older than 8 weeks, and all were at least 6 weeks old. The labeling observed was comparable in utricles from older and younger specimens. All protocols were in accordance with the University of Virginia's guidelines for use of animals in research. 5. Cultures were incubated for 24 hours in Medium-199 with 20% FBS that contained 0.5 to 1.0 mM neomycin or 1.0 to 2.0 mM gentamicin. 6. After 24 hours, cultures were rinsed twice with Medium-199, and fresh medium without any aminoglycoside antibiotics was added. The aminoglycoside-free media contained either of two mitotic tracers: [3H]methyl-thymidine (0.8 gCi/ml, 65Ci/mmol) or 5-bromo-2'-deoxyuridine (BrdU, 3 gg/ml) in solution with 5-fluoro-2'-deoxyuridine (0.27 gg/ml) from Amersham.

The transcription factors E2A (E12/E47) and Pip are both required for normal B-cell development. Each protein binds to regulatory sequences within various immunoglobulin enhancer elements. Activity of E2A proteins can be regulated by interactions with other proteins which influence their DNA binding or activation potential. Similarly, Pip function can be influenced by interaction with the protein PU.1, which can recruit Pip to bind to DNA. We show here that a previously unidentified Pip binding site resides adjacent to the E2A binding site within the immunoglobulin 3 enhancer. Both of these binding sites are crucial for high-level enhancer activity. We found that E47 and Pip can functionally interact to generate a very potent 100-fold transcriptional synergy. Through a series of mutagenesis experiments, we identified the Pip sequences necessary for transcriptional activation and for synergy with E47. Two synergy domains (residues 140 to 207 and 300 to 420) in addition to the Pip DNA binding domain (residues 1 to 134) are required for maximal synergy with E47. We also identified a Pip domain (residues 207 to 300) that appears to mask Pip transactivation potential. Part of the synergy mechanism between E47 and Pip appears to involve the ability of Pip to increase DNA binding by E47, perhaps by inducing a conformational change in the E47 protein. E47 may also induce a conformational change in Pip which unmasks sequences important for transcriptional activity. Based upon our results, we propose a model for E47-Pip transcriptional synergy.

The methods developed in this study can be applied to screen for genes capable of inducing an OA-like phenotype in chondrocytes on a genome-wide scale and identify novel mediators of OA pathogenesis. Thus, coordinated functional genomic approaches can be used to delineate key genes and pathways activated in complex human diseases such as OA.

E47 and Pip are proteins crucial for proper B-cell development. E47 and Pip cooperatively bind to adjacent sites in the immunoglobulin kappa chain 3 enhancer and generate a potent transcriptional synergy. We generated protein-DNA computer models to visualize E47 and Pip bound to DNA. These models predict precise interactions between the two proteins. We tested predictions deduced from these models by mutagenesis studies and found evidence for novel direct interactions between the E47 helix-loop-helix domain (Arg 357 or Asp 358) and the Pip N terminus (Leu 24). We also found that precise spatial alignment of the binding sites was necessary for transcriptional synergy and cooperative DNA binding. A Pip dominant negative mutant that cannot synergize with E47 inhibited enhancer activity in plasmacytoma cells and could not activate transcription in pre-B cells. Using electrophoretic mobility shift assays, we found that Pip can bind to the heavy-chain intron enhancer region. In addition, we found that in fibroblasts Pip greatly increased E47 induction of germ line I transcripts associated with somatic rearrangement and isotype class switching. However, a Pip dominant negative mutant inhibited germ line I transcripts. The importance of these results for late B-cell functions is discussed.During B-cell development, cells progress through an ordered series of steps, including pro-B-, pre-B-, B-, and plasmacell stages. These stages can be defined by expression of specific cell surface markers and ordered rearrangement of immunoglobulin (Ig) heavy-chain and light-chain genes (28). The heavy-chain genes usually rearrange first, early in B-cell development during the change from the pro-B-to the pre-Bcell stage. Ig light-chain genes (kappa and lambda) are unrearranged and transcriptionally silent at the pro-B-cell stage but undergo somatic rearrangement during the pre-B-to B-cell transition to produce a productive light-chain gene. B cells subsequently undergo class switch recombination to produce antibodies with different effector functions.A variety of studies indicate that enhancers at the Ig heavychain and light-chain loci are very important for proper B-cell development (23,59,63). These enhancers play crucial roles not only in Ig transcription but also in somatic rearrangement, isotype class switch recombination, somatic mutation, and control of chromatin structure (5,35,37,43,48). A variety of transcription factors [E2A, EBF, PU