Interallelic Class Switch Recombination Contributes Significantly to Class Switching in Mouse B Cells (original) (raw)
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Immunity, 2001
proteins, which altogether may either mediate repres-CNRS UMR6101 sion of the 3Ј elements in lymphocytes or activation in Faculté de Mé decine plasma cells (Muto et al., 1998). By contrast, the distant Limoges hs4 enhancer, lying some 26 kb downstream of the 2 Centre de Dé veloppement des Techniques Avancé es ␣ gene, seems to be active throughout B cell differentiapour l'Expé rimentation Animale tion (Michaelson et al., 1995). Its activity in pre-B cells UPS CNRS 44 may rely on the presence of oct and B sites; BSAP Orlé ans binds hs4 in a differential manner, apparently repressing France the enhancer in pre-B cells and boosting its activity in mature B cells (Michaelson et al., 1996). Based on transient transfection experiments, it was Summary concluded that the four enhancers are rather weak when they individually drive transcription of reporter genes. Four transcriptional enhancers lie downstream of the However, their combinations displayed strong transcripimmunoglobulin heavy chain locus: C␣3/hs3a, hs1,2,
Enhancer–promoter communication and transcriptional regulation of Igh
Trends in Immunology, 2011
Transcriptional regulation of eukaryotic protein-coding genes requires the participation of sitespecific transcription factors that bind distal regulatory elements, as well as factors which, together with RNA polymerase II, form the basal transcription machinery at the core promoter. Because gene regulation requires proper communication between promoters and enhancers, often over great distances, it is important to understand the potentially interrelated transcription factor interactions at both of these elements. How this is achieved on tissue-specific genes, such as the immunoglobulin heavy chain (IgH) in B cells remains unclear. Here we review known interactions at the Igh variable region (V H) promoters and present our perspective on promoter-enhancer interactions that are likely important for Ig gene regulation in B cells. Regulated expression of Igh The seminal studies of Jacob and Monod performed in E. Coli led to the idea of regulated transcription (1). Subsequent biochemical and genetic analyses established key aspects of gene regulation in prokaryotes (2). It has, however, been far more challenging to obtain a comparable understanding of the mechanisms that underlie tissue-specific gene regulation in mammals. Beyond the increased complexity of mammalian genomes and transcriptional machineries (3), this also reflects the difficulty of faithfully recapitulating, in well-defined in vitro systems, the long-range enhancer-promoter interactions that are necessary for tissue specific gene expression. While this has somewhat hampered our understanding of mechanistic details of regulatory processes, significant knowledge about tissue-specific transcriptional regulation has been derived from transgenic studies in murine models as well as cell-based transfection and/or transduction studies. Collectively, these studies have revealed that site-specific, enhancer-bound regulatory factors communicate, through various cofactors, with the basal transcriptional machinery that associates with core promoters near the transcriptional initiation site (3). However, for most genes the precise molecular mechanisms and contact points between the enhancer-and promoter-bound transcription factors remain unclear. Added to this complexity is the fact that tissue-specific genes are subject to natural constraints that often involve regulation of chromatin structure and subnuclear positioning (4-7). For instance, Ig genes have to negotiate tissue-and
The Igκ 3′ Enhancer Influences the Ratio of Igκ versus Igλ B Lymphocytes
Immunity, 1996
Nienke van der Stoep,* al., 1980; reviewed by Chen et al., 1994).-producing cells often lack one or both C exons and 3Ј C se-Robert Monroe,* Michel Cogne,* § quences owing to a V(D)J recombinational event involv-Laurie Davidson,* † and Frederick W. Alt* † ‡ ing a recombination signal sequence (termed RS in mice) *Howard Hughes Medical Institute located about 25 kb 3Ј of the C exon, suggesting a The Children's Hospital potential role for this process in promoting re-† Department of Genetics arrangements (reviewed by Selsing and Daitch, 1995). Harvard Medical School While rearrangement of the RS sequences themselves ‡ Center for Blood Research is not required for gene rearrangement (Chen et al., Boston, Massachusetts 02115 1993b; Takeda et al., 1993; Zou et al., 1993), RS rearrangements might increase the generation of producers by other mechanisms, for example by deletion Summary of elements within the locus that inhibit rearrangement or by increasing the frequency of cells with non-We generated mice harboring germline mutations in functional genes (reviewed by Selsing and Daitch, which the enhancer element located 9 kb 3 of the 1995). immunoglobulin light chain gene (3E) was replaced Individual B cells express only one of their two allelic either by a single loxP site (3E ⌬) or by a neomycin H chain loci and one of their multiple L chain loci as resistance gene (3E N). Mice homozygous for the proteins that associate to form complete immunoglobu-3E ⌬ mutation had substantially reduced numbers lin (reviewed by Lansford et al., 1996). These allelic and of-expressing B cells and increased numbers of isotype (versus) exclusion processes are effected-expressing B cells accompanied by decreased verby feedback mechanisms; for example, functional L sus gene rearrangement. In these mutant mice, chain expression curtails further gene rearrangement expression was reduced in resting B cells, but was and eliminates the opportunity for gene rearrangement normal in activated B cells. The homozygous 3E N to be initiated (Alt et al., 1980; Ritchie et al., 1984). The mutation resulted in a similar but more pronounced ratio of to B cells (/ ratio) in mice and humans phenotype. Both mutations acted in cis. These studies (about 20/1 and 2/1, respectively) correlates with the show that the 3E is critical for establishing the normal relative complexity of the V to V loci in those species / ratio, but is not absolutely essential for gene (reviewed by Lansford et al., 1996). As gene rerearrangement or, surprisingly, for normal expresarrangement occurs preferentially to that of genes in sion in activated B cells. These studies also imply the both species (Coleclough et al., 1981; Korsmeyer et al., existence of additional regulatory elements that have 1982), the relative / ratios may be influenced by postoverlapping function with the 3E element. rearrangement mechanisms (Langman and Cohn, 1995). All antigen receptor loci contain tissue-specific and Introduction developmentally regulated enhancer elements (reviewed by Calame and Ghosh, 1995). The Ig locus has Immunoglobulin (Ig) heavy (H) and light (L) chain variable two known enhancers: the intronic enhancer (iE), region genes are assembled from multiple germline V, which lies between J and C (Emorine et al., 1983; Par-(D), and J gene segments during early B cell differentiaslow and Granner, 1982; Picard and Schaffner, 1984), tion (reviewed by Lansford et al., 1996; Okada and Alt, and the 3Ј enhancer (3ЈE), which lies about 9 kb down-1995). The variable region gene segments in the single stream of C (Meyer and Neuberger, 1989). Most infor-IgH chain locus and two IgL chain loci (and) are mation regarding potential functions of the enhancers assembled by a common V(D)J recombinase. Recomes from assays of transfected and transgenic exarrangement and expression of IgH chain variable region pression constructs. The iE becomes active during the genes usually precedes that of IgL chain genes (repre-B to B cell transition and may function in activating viewed by Lansford et al., 1996). IgL chain gene relocus recombination and transcription (reviewed by arrangement also appears ordered; it usually initiates Calame and Ghosh, 1995). The 3ЈE is active at the B-cell at the locus, with gene rearrangements occurring stage and beyond (Meyer and Neuberger, 1989; Park predominantly in cells that have attempted and failed and Atchison, 1991; Pongubala and Atchison, 1991). to assemble productive genes (Alt et al., 1980; Cole-Studies of transgenic loci suggested that 3ЈE is reclough et al., 1981; Korsmeyer et al., 1982). quired for high level expression in immunoglobulin-The apparent ordering of L chain gene rearrangement secreting cells and for efficient exclusion of endogenous might result from either a specific mechanism that ef-L chain gene rearrangement (Betz et al., 1994; Meyer et fects "ordered" activation of the and loci or a "stoal., 1990). Transfection studies further indicated that 3ЈE chastic" mechanism in which these loci are activated and iE may function synergistically to promote transimultaneously, with the probability of gene rescription in B cell lines (Fulton and Van Ness, 1993, arrangement greatly exceeding that of genes (Alt et 1994). Enhancer elements promote rearrangement of linked V, (D), and J segments in transfected and transgenic
The murine Ig heavy chain (IgH) 3 regulatory region contains four enhancers: hs3A, hs1,2, hs3B, and hs4. Various studies have suggested a role for these enhancers in regulating IgH expression and class switching. Here we assess the role of hs3A and hs1,2 in these processes by exploiting a naturally occurring deletion of these enhancers from the expressed, C57BL/6 allele of the F 1 pre-B cell line, 70Z/3. Equivalent expression in 70Z/3 and 18-81 (which has an intact 3 region) indicated that hs3A and hs1,2 were not essential for expression at the pre-B cell stage. To further examine the role of hs3A and hs1,2 in IgH function at the plasma cell stage, we fused 70Z/3 with the plasmacytoma NSO. Electromobility shift assay analysis of the 70Z/3-NSO hybrids revealed a transcription factor complement conducive to the activation of the 3 enhancers. Despite the lack of enhancers, hs3A and hs1,2, the level of RNA and protein in the 70Z/3-NSO fusion hybrids was substantially elevated relative to its pre-B parent and comparable with that observed in a number of -producing spleen cell hybridomas. Additionally, ELISAspot assays showed that the 70Z/3-NSO hybrid underwent spontaneous class switching in culture to IgG1 at a frequency comparable with that of most hybridomas. These results indicate that hs3A and hs1,2 are not essential for high levels of IgH expression or for spontaneous class switching in a plasma cell line. Abbreviations used in this paper: IgH, immunoglobulin heavy chain protein; Igh, immunoglobulin heavy chain gene; V H , heavy chain variable region gene; C H , heavy chain constant region gene; hs, DNase I hypersensitive site; LCR, locus control region; EMSA, electromobility shift assay; BSAP, B cell-specific activator protein (Pax5); NP, (4-hydroxy-3-nitrophenyl)acetyl.
The EMBO journal, 1992
Antibody class switching is achieved by recombinations between switch (S) regions which consist of tandemly repeated sequences located 5' to Ig heavy chain constant (CH) region genes. RNA transcripts from specific unrearranged or germ-line Ig CH genes are induced in IgM+ B cells prior to their undergoing class switch recombination to the same CH genes. Thus, the antibody class switch appears to be directed by induction of accessibility, as assayed by transcription of germ line CH genes. For example, IL-4 induces transcripts from the mouse germ-line C gamma 1 and C epsilon genes to which it also directs switch recombination. We report here that the 150 bp region upstream of the first initiation site of RNA transcribed from the murine germ-line C gamma 1 gene, contains promoter and enhancer elements responsible for basal level transcription and inducibility by anti-Ig phorbol myristate acetate (PMA) and for synergy of these inducers with IL-4 in a surface IgM+ B cell line, L10A6.2...