The assembly of Ag receptor genes by V(D)J recombination is regulated by transcriptional promoters and enhancers which control chromatin accessibility at Ig and TCR gene segments to the RAG-1/RAG-2 recombinase complex. Paradoxically, germline deletions of the IgH enhancer (Eμ) only modestly reduce DH→JH rearrangements when assessed in peripheral B cells. However, deletion of Eμ severely impairs recombination of VH gene segments, which are located over 100 kb away. We now test two alternative explanations for the minimal effect of Eμ deletions on primary DH→JH rearrangement: 1) Accessibility at the DHJH cluster is controlled by a redundant cis-element in the absence of Eμ. One candidate for this element lies 5′ to DQ52 (PDQ52) and exhibits promoter/enhancer activity in pre-B cells. 2) In contrast to endpoint B cells, DH→JH recombination may be significantly impaired in pro-B cells from enhancer-deficient mice. To elucidate the roles of PDQ52 and Eμ in the regulation of IgH locus accessibility, we generated mice with targeted deletions of these elements. We report that the defined PDQ52 promoter is dispensable for germline transcription and recombination of the DHJH cluster. In contrast, we demonstrate that Eμ directly regulates accessibility of the DHJH region. These findings reveal a significant role for Eμ in the control mechanisms that activate IgH gene assembly and suggest that impaired VH→DHJH rearrangement in enhancer-deficient cells may be a downstream consequence of the primary block in DH→JH recombination.

V(D)J recombination is believed to be regulated by alterations in chromatin accessibility to the recombinase machinery, but the mechanisms responsible remain unclear. We previously proposed that antisense intergenic transcription, activated throughout the mouse Igh V H region in pro-B cells, remodels chromatin for V H -to-DJ H recombination. Using RNA fluorescence in situ hybridization, we now show that antisense intergenic transcription occurs throughout the Igh D H J H region before D-to-J recombination, indicating that this is a widespread process in V(D)J recombination. Transcription initiates near the Igh intronic enhancer E and is abrogated in mice lacking this enhancer, indicating that E regulates D H antisense transcription. E was recently demonstrated to regulate D H -to-J H recombination of the Igh locus. Together, these data suggest that E controls D H -to-J H recombination by activating this form of germ line Igh transcription, thus providing a long-range, processive mechanism by which E can regulate chromatin accessibility throughout the D H region. In contrast, E deletion has no effect on V H antisense intergenic transcription, which is rarely associated with D H antisense transcription, suggesting differential regulation and separate roles for these processes at sequential stages of V(D)J recombination. These results support a directive role for antisense intergenic transcription in enabling access to the recombination machinery.In order to generate the primary repertoire of immunoglobulin (Ig) and T-cell receptor (TCR) molecules, antigen receptor loci undergo variable, diversity, and joining [V(D)J] recombination in B and T lymphocytes. Recombination is catalyzed by a recombinase complex containing the protein products of the recombinase-activating genes Rag1 and Rag2 (28). Within precursor lymphocytes, this process is strictly lineage specific, with heavy (Igh) and light (Ig and Ig) immunoglobulin loci fully recombining only in B lymphocytes and T-cell receptor loci (Tcra, Tcrb, Tcrg, and Tcrd) recombining only in T cells. Further, within lineages, loci are recombined in a precise order. Recombination of the Igh locus is the earliest step in the generation of the mature antibody repertoire in B lymphocytes. The Igh locus of the C57BL/6 mouse spans 3 Mb and comprises 195 V H genes spanning 2.5 Mb, 10 D H genes (ϳ60 kb), 4 J H genes (2 kb), and 8 constant (C H ) genes (200 kb) (31, 68). D H -to-J H recombination occurs on both Igh alleles before V H -to-DJ H recombination takes place (16).Lineage and stage specificity of V(D)J recombination are regulated by differential chromatin accessibility to the RAG proteins. Several mechanisms may contribute, but their relative importance is still unclear. The first process discovered was germ line transcription, which occurs in all antigen receptor loci across gene segments competent for recombination (34).This transcription was termed "sterile" or "germ line" to distinguish it from coding V(D)J transcription. In the Igh locus, the earliest germ line transc...

Nuclear factor B (NF-B) and the Rel family of proteins are pleiotropic transcription factors that play central roles in the immune and inflammatory responses, as well as apoptosis. Here, we identified a serine/threonine protein phosphatase X (PPX; also called protein phosphatase 4 (PP4)) that specifically associated with c-Rel, NF-B p50, and RelA. The amino acid sequences of human and mouse PPX are 100% identical, and the PPX gene was mapped to human chromosome 16 p11.2. Overexpression of PPX, but not catalytically inactive PPX mutants, stimulated the DNA-binding activity of c-Rel and activated NF-B-mediated transcription. These results suggest that PPX is a novel activator of c-Rel/NF-B.The Rel/NF-B 1 family of transcription factors includes RelA (also called NF-B p65), RelB, c-Rel, NF-B1 p50 (also called NF-B p50), and NF-B2 p52 and is involved in immunological responses, cellular proliferation, and programmed cell death (1, 2). Rel/NF-B family members share the 300-amino acid Rel homology domain in their amino-terminal regions. They activate gene expression by binding to B sites via the DNAbinding domain located within this Rel homology region. Although the regulation of Rel/NF-B by its inhibitor IB has been extensively studied, control of the phosphorylation state of Rel/NF-B remains unclear. Previously, we showed that c-Rel and RelA are involved in CD28-mediated signal transduction (3, 4). We also showed that c-Rel is both constitutively phosphorylated in T cells and that it is also inducibly phosphorylated following T-cell receptor plus CD28 costimulation in T cells (3). c-Rel knockout mice exhibit profound defects in T-cell function, including lymphokine (interleukin 2, interleukin 3, and granulocyte macrophage-colony stimulating factor) secretion and the T-cell proliferative response to T-cell receptor plus CD28 costimulation (1). To further study the signaling pathway leading to c-Rel activation, we searched for c-Rel interacting kinases or phosphatases using the yeast two-hybrid system. Here we identify the serine/threonine protein phosphatase X (PPX; also called protein phosphatase 4 (PP4)) that specifically associated with c-Rel and activated NF-B-mediated transcription. MATERIALS AND METHODSYeast Two-hybrid Screening-Yeast single-copy plasmids pPC62 and pPC86 (6) were kindly provided by Dr. D. Nathans; the yeast-selectable marker genes LEU2 and TRP1 were swapped between these two plasmids, resulting in a GAL4 transcriptional activation domain vector containing the LEU2 selectable marker (designated pTA), and a GAL4 DNA-binding domain vector carrying the TRP1 selectable marker (designated pDB). DNA encoding amino acids 2 to 300 of c-Rel was cloned into the GAL4 DNA-binding domain vector pDB, designated pDB-c-Rel, and used as bait to screen a human B lymphocyte cDNA library (CLON-TECH). Positive yeast clones were selected by colony filter ␤-galactosidase assay as described (6), and yeast DNA was recovered from positive clones and transformed into Escherichia coli. Plasmids containing cDNA clones were ...