The murine Igh locus has a 3 regulatory region (3 RR) containing four enhancers (hs3A, hs1,2, hs3B, and hs4) at DNase I-hypersensitive sites. The 3 RR exerts long-range effects on class switch recombination (CSR) to several isotypes through its control of germ line transcription. By measuring levels of acetylated histones H3 and H4 and of dimethylated H3 (K4) with chromatin immunoprecipitation assays, we found that early in B-cell development, chromatin encompassing the enhancers of the 3 RR began to attain stepwise modifications typical of an open conformation. The hs4 enhancer was associated with active chromatin initially in pro-and pre-B cells and then together with hs3A, hs1,2, and hs3B in B and plasma cells. The immunoglobulin heavy chain locus (Igh) (see Fig. 1) is arguably the most complex mammalian locus. B-cell development is marked by the progressive expression of Igh genes (via VDJ recombination, somatic hypermutation, and class switch recombination [CSR]). These regulated processes, some involving substantial DNA rearrangements and deletions, are required for the generation of antibody diversity (reviewed in references 47 and 57). Spanning ϳ3 Mb, the murine Igh locus contains a limited number of enhancers, including the intronic enhancer (E), which has been implicated in V to DJ joining and chain expression (reviewed in reference 47), and a complex 3Ј regulatory region (3Ј RR) located immediately downstream of C␣, the last constant region gene. The human Igh locus contains two 3Ј RRs, each located downstream of one of the duplicated C␣ genes (9, 49, 62).The murine 3Ј RR spans ϳ28 kb downstream of C␣ and comprises four enhancers, each associated with DNase I hypersensitivity (hs3A, hs1,2, hs3B, and hs4) (reviewed in reference 33). hs3A and hs3B are located at the termini of a 25-kb inverted repeat flanking hs1,2 (7, 66), and these three enhancers acquire DNase I hypersensitivity at later stages of B-cell development (66). The hs4 enhancer is the only 3Ј enhancer with activity and DNase I hypersensitivity throughout B-cell development (19, 48), although no role for hs4 or any of the 3Ј enhancers in early B-cell development has been shown.Targeted deletions of the 3Ј RR enhancers have shown that hs3A and hs1,2 are individually dispensable for B-cell development and Igh expression (46), while the combination of hs3B and hs4 is critical for the process of CSR (63). The 3Ј RR has also been proposed to regulate Igh expression in plasma cells (23,40,48,63,70). In accord with earlier suggestions that it functions as a locus control region (43), the 3Ј RR shows synergistic activity and position-independent regulation when tested in transgenic model systems (8,33,59). However, copynumber-dependent expression has not been observed (8). In addition to a function within the Igh locus, the 3Ј RR is likely to be responsible for dysregulation of c-myc expression resulting from certain Igh::c-myc translocations in mouse plasmacytoma cells as well as in human Burkitt's lymphoma and multiple myeloma cells (30,36,43).Th...
A tissue-specific enhancer (E mu) lies between the joining (JH) and mu constant region (C mu) gene segments of the immunoglobulin heavy chain (IgH) locus. Since mouse endogenous IgH genes are efficiently transcribed in its absence, the normal function of this enhancer remains ill-defined. Recently, another lymphoid-specific enhancer of equal strength has been identified 3' of the rat IgH locus. We have isolated an analogous sequence from mouse and have mapped it 12.5 kb 3' of the 3'-most constant region gene (C alpha-membrane) of the BALB/c mouse locus. The mouse and rat sequences are 82% homologous and share with other enhancers several DNA sequence motifs capable of binding protein. However, in transient transfection assays, the mouse sequence behaves as a weaker enhancer. The role of this distant element in the expression of endogenous IgH genes, both in E mu-deficient, Ig-producing cell lines and during normal B cell development, is discussed.
The E2A proteins, E12 and E47, are basic helix-loophelix (bHLH) proteins essential for the B-cell lineage. Initially identified as immunoglobulin enhancer-binding proteins, they have also been shown to activate immunoglobulin enhancer-based reporters in transient transfection assays. Here, we examine the relationship between E2A DNA binding activity and activation of the endogenous, chromosomal immunoglobulin heavy chain (IgH) locus. Using sterile Ii transcription as an indicator of IgH enhancer activity, we see a direct correlation between E2A DNA binding activity and Ij transcription in stable BXT hybrids. We also observe a 1000-fold stimulation of endogenous Ij transcription in fibroblasts that express high levels of E47 and less stimulation in cells that express E12. By contrast, none of the other IgH enhancer-binding proteins tested (E2-2, Pu.1, Oct-2, OCA-B, TFE3 and USF) were able to activate IF transcription. E47 overexpression also resulted in transcriptional activation of the endogenous gene encoding TdT, indicating that it, too, is a target of E2A proteins early in the B-cell lineage. Our results indicate that E2A proteins have the distinctive property of activating silent, chromatin-embedded B-cellspecific genes, underscoring their crucial role in B-cell development. Keywords: B lymphocytes/E2A proteins/E47/ immunoglobulin/Td/transcription 5014 C Oxford University Press E47 activates IgH and TdT loci in non-B cells C J J J -4. i J2J 3 J 'ef~~-
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