The immunoglobulin light-chain gene enhancers EK3,, Ex2.4, and E~3. l contain a conserved cell type-specific composite element essential for their activities. This element binds a B cell-specific heterodimeric protein complex that consists of the Ets family member PU.1 and a second factor (NF-EMS), whose participation in the formation of the complex is dependent on the presence of DNA-bound PU.1. In this report we describe the cloning and characterization of Pip (_PU.I "_interaction partner), a lymphoid-specific protein that is most likely NF-EMS. As expected, the Pip protein binds the composite element only in the presence of PU.I; furthermore, the formation of this ternary complex is critically dependent on phosphorylation of PU.1 at serine-148. The P/p gene is expressed specifically in lymphoid tissues in both B-and T-cell lines. When coexpressed in NIH-3T3 cells, Pip and PU.1 function as mutually dependent transcription activators of the composite element. The amino-terminal DNA-binding domain of Pip exhibits a high degree of homology to the DNA-binding domains of members of the interferon regulatory factor (IRF) family, which includes IRF-1, IRF-2, ICSBP, and ISGF3~/.

Immunoglobulin (Ig) genes are hypermutated in B lymphocytes that are the precursors to memory B cells. The mutations are linked to transcription initiation, but non-Ig promoters are permissible for the mutation process; thus, other genes expressed in mutating B cells may also be subject to somatic hypermutation. Significant mutations were not observed in c-MYC, S14, or alpha-fetoprotein (AFP) genes, but BCL-6 was highly mutated in a large proportion of memory B cells of normal individuals. The mutation pattern was similar to that of Ig genes.

To identify DNA sequences that target the somatic hypermutation process, the immunoglobulin gene promoter located upstream of the variable (V) region was duplicated upstream of the constant (C) region of a kappa transgene. Normally, kappa genes are somatically mutated only in the VJ region, but not in the C region. In B cell hybridomas from mice with this kappa transgene (P5'C), both the VJ region and the C region, but not the region between them, were mutated at similar frequencies, suggesting that the mutation mechanism is related to transcription. The downstream promoter was not occluded by transcripts from the upstream promoter. In fact, the levels of transcripts originating from the two promoters were similar, supporting a mutation model based on initiation of transcripts. Several "hot-spots" of somatic mutation were noted, further demonstrating that this transgene has the hallmarks of somatic mutation of endogenous immunoglobulin genes. A model linking somatic mutation to transcription-coupled DNA repair is proposed.

Pip is a lymphoid-restricted IRF transcription factor that is recruited to composite elements within immunoglobulin light-chain gene enhancers through a specific interaction with the Ets factor PU.I. We have examined the transcriptional regulatory properties of Pip as well as the requirements for its interaction with PU.1 and DNA to form a ternary complex. We demonstrate that Pip is a dichotomous regulator; it specifically stimulates transcription in conjunction with PU.1, but represses odl3-interferon-inducible transcription in the absence of PU.1. Thus, during B-cell activation and differentiation, Pip may function both as an activator to promote B cell-specific gene expression and as a repressor to inhibit the antiproliferative effects of a/13-interferons. Mutational analysis of Pip reveals a carboxy-terminal segment that is important for autoinhibition of DNA binding and ternary complex formation. A domain of Pip containing this segment confers autoinhibition and PU.l-dependent binding activity to the DNA-binding domain of the related IRF family member, p48. On the basis of these and other data we propose a model for PU.1/Pip ternary complex formation.

B-cell-specific enhancers have been identified in the immunoglobulin K locus 3' of each constant-region cluster. These enhancers contain two distinct domains, AA and AB, which are essential for enhancer function.AB contains a near-consensus binding site for the Ets family of transcription factors. In this study, we have identified a B-cell-specific protein complex which binds the AB motif of the A2-4 enhancer in vitro and appears necessary for the activity of the enhancer in vivo, since mutations in AB which prevent this interaction also eliminate enhancer function. This complex contains PUA, a member of the Ets family, and a transcriptional activator whose expression is restricted to cells of the hematopoietic system with the exception of T lymphocytes. In addition, it contains a factor which binds specifically to a region adjacent to the PU.1 binding site. This factor cannot bind AB autonomously but appears to require interaction with the PU.1 protein to stabilize its association with the DNA. This complex may be identical or related to the PU.1/NF-EM5 complex which interacts with a homologous DNA element in the immunoglobulin K 3' enhancer.The expression of murine immunoglobulin (Ig) heavy-and light-chain genes is tightly controlled in a cell-type-and developmental stage-specific fashion. This regulation occurs at two levels, transcription and recombination. Transcription of Ig genes is regulated by cell-type-specific promoter and enhancer elements (8,59,62). In addition, a lymphocyte-specific recombinase acts in a temporally regulated manner to recombine the germ line Ig loci, resulting in the creation of open reading frames which code for functional heavy-and light-chain protein molecules (13, 56). It has been suggested that the binding of regulatory factors to cis-acting transcriptional control regions may precede Ig gene rearrangement and may in fact effect the changes in the chromatin structure of these regions which allow access of the recombinase machinery (5,14,57,70). In this manner, control of Ig gene rearrangement may be mediated by the tissue-specific and developmentally regulated expression of trans-acting promoter-and enhancer-binding factors in B cells. Therefore, enhancer and promoter elements may play an important role in the initiation of the recombination process as well as in the cell-type-and stage-specific control of the expression of functionally rearranged Ig genes.The control elements which regulate Ig gene expression have been extensively studied. B-cell-specific enhancers have been identified and well characterized in the major J-C introns of both the heavy (2, 18, 38) and K light-chain (46, 51) genes. These enhancers are modular in structure, consisting of multiple, often redundant binding sites for positive-and negative-acting nuclear factors. Some of these sites are shared among enhancers, while others are unique to a particular enhancer and may thus confer specificity of function. Complex protein-protein interactions and subtle variations in factor concentrations may affect enha...