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...

The activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class-switch recombination of Ig genes. It has been shown that in vitro, AID protein deaminates C in single-stranded DNA or the coding-strand DNA that is being transcribed but not in double-stranded DNA. However, in vivo, both DNA strands are mutated equally during SHM. We show that AID efficiently deaminates C on both DNA strands of a supercoiled plasmid, acting preferentially on SHM hotspot motifs. However, this DNA is not targeted by AID when it is relaxed after treatment with topoisomerase I, and thus, supercoiling plays a crucial role for AID targeting to this DNA. Most of the mutations are in negatively supercoiled regions, suggesting a mechanism of AID targeting in vivo. During transcription the DNA sequences upstream of the elongating RNA polymerase are negatively supercoiled, and this transient change in DNA topology may allow AID to access both DNA strands.A major advance in the study of somatic hypermutation (SHM) and class-switch recombination (CSR) has been the discovery of the activation-induced cytidine deaminase (AID) (1-3), which appears to be the long-sought SHM mutator factor and inducer of CSR (4). Current in vitro data show that AID is a DNA-specific cytidine deaminase that preferentially removes the amino group of cytidine in single-stranded DNA and in the nontranscribed strand when transcription is active (5-11). These findings are consistent with previous experiments in which SHM is linked to transcription (12, 13). However, in vivo, both DNA strands are equally mutated (14). Furthermore, in ung Ϫ/Ϫ mice, where almost all of the C or G mutations are transitions due to unrepaired AID lesions, equal targeting of both strands is confirmed (15). Single-stranded DNA would also occur in vivo as a potential AID target during DNA replication, which would explain why both DNA strands are targeted during SHM and CSR. However, experiments with a cell line that undergoes SHM in culture support the conclusion that AID can act during the G 1 phase of the cell cycle, and therefore, is not restricted to the S phase (16) (S. Gasior and U.S., unpublished data). A nonexclusive third possibility is that DNA topology (e.g., supercoiling) creates an AID-accessible conformation. In vivo, Ig genes are associated in nucleosomes with histones and other chromatin proteins. These associations, as well as the process of transcription, affect the topology of DNA. To test the possibility that supercoiled DNA as it exists in vivo may be a target for AID, we carried out cytidine deamination assays in vitro with AID purified from insect cells (5). The supercoiled target DNA was an Escherichia coli plasmid that had been manipulated to allow bacterial resistance to carbenicillin only when the initiator AUG of an ampicillin resistance (Amp r ) gene was created by AID deamination of an ACG triplet. Materials and MethodsPlasmid Construction. The kanamycin-resistance (Kan r ) gene was inserted into the SacII site in pBluescript KS(II...

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Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by activation-induced cytosine deaminase (AID). The uracil, and potentially neighboring bases, are processed by error-prone base excision repair and mismatch repair. Deficiencies in Ung, Msh2, or Msh6 affect SHM and CSR. To determine whether Msh2/Msh6 complexes which recognize single-base mismatches and loops were the only mismatch-recognition complexes required for SHM and CSR, we analyzed these processes in Msh6−/−Ung−/− mice. SHM and CSR were affected in the same degree and fashion as in Msh2−/−Ung−/− mice; mutations were mostly C,G transitions and CSR was greatly reduced, making Msh2/Msh3 contributions unlikely. Inactivating Ung alone reduced mutations from A and T, suggesting that, depending on the DNA sequence, varying proportions of A,T mutations arise by error-prone long-patch base excision repair. Further, in Msh6−/−Ung−/− mice the 5′ end and the 3′ region of Ig genes was spared from mutations as in wild-type mice, confirming that AID does not act in these regions. Finally, because in the absence of both Ung and Msh6, transition mutations from C and G likely are “footprints” of AID, the data show that the activity of AID is restricted drastically in vivo compared with AID in cell-free assays.