A novel pax5-binding regulatory element in the ig? locus.
ABSTRACT: The Ig? locus undergoes a variety of different molecular processes during B cell development, including V(D)J rearrangement and somatic hypermutations (SHM), which are influenced by cis regulatory regions (RRs) within the locus. The Ig? locus includes three characterized RRs termed the intronic (iE?), 3'E?, and Ed enhancers. We had previously noted that a region of DNA upstream of the iE? and matrix attachment region (MAR) was necessary for demethylation of the locus in cell culture. In this study, we further characterized this region, which we have termed Dm, for demethylation element. Pre-rearranged Ig? transgenes containing a deletion of the entire Dm region, or of a Pax5-binding site within the region, fail to undergo efficient CpG demethylation in mature B cells in vivo. Furthermore, we generated mice with a deletion of the full Dm region at the endogenous Ig? locus. The most prominent phenotype of these mice is reduced SHM in germinal center B cells in Peyer's patches. In conclusion, we propose the Dm element as a novel Pax5-binding cis regulatory element, which works in concert with the known enhancers, and plays a role in Ig? demethylation and SHM.
Project description:Somatic hypermutation (SHM) diversifies the V region of Ig genes and underlies the process of affinity maturation, in which B lymphocytes producing high-affinity Abs are generated and selected. SHM is triggered in activated B cells by deamination of deoxycytosine residues mediated by activation-induced deaminase (AID). Whereas mistargeting of SHM and AID results in mutations and DNA damage in many non-Ig genes, they act preferentially at Ig loci. The mechanisms responsible for preferential targeting of SHM and AID activity to Ig loci are poorly understood. Using an assay involving an SHM reporter cassette inserted into the Ig L chain locus (IgL) of chicken DT40 B cells, we have identified a 1.9-kb DIVAC (diversification activator) element derived from chicken IgL that supports high levels of AID-dependent mutation activity. Systematic deletion analysis reveals that targeting activity is spread throughout much of the sequence and identifies two core regions that are particularly critical for function: a 200-bp region within the IgL enhancer, and a 350-bp 3' element. Chromatin immunoprecipitation experiments demonstrate that whereas DIVAC does not alter levels of several epigenetic marks in the mutation cassette, it does increase levels of serine-5 phosphorylated RNA polymerase II in the mutation target region, consistent with an effect on transcriptional elongation/pausing. We propose that multiple, dispersed DNA elements collaborate to recruit and activate the mutational machinery at Ig gene variable regions during SHM.
Project description:Interactions with cognate antigens recruit activated B cells into germinal centers where they undergo somatic hypermutation (SHM) in V(D)J exons for the generation of high-affinity antibodies. The contribution of IgH transcriptional enhancers in SHM is unclear. The E? enhancer upstream of C? has a marginal role, whereas the influence of the IgH 3' regulatory region (3'RR) enhancers (hs3a, hs1,2, hs3b, and hs4) is controversial. To clarify the latter issue, we analyzed mice lacking the whole 30-kb extent of the IgH 3'RR. We show that SHM in VH rearranged regions is almost totally abrogated in 3'RR-deficient mice, whereas the simultaneous Ig heavy chain transcription rate is only partially reduced. In contrast, SHM in ? light chain genes remains unaltered, acquitting for any global SHM defect in our model. Beyond class switch recombination, the IgH 3'RR is a central element that controls heavy chain accessibility to activation-induced deaminase modifications including SHM.
Project description:Somatic hypermutation (SHM) introduces point mutations into immunoglobulin (Ig) genes but also causes mutations in other parts of the genome. We have used lentiviral SHM reporter vectors to identify regions of the genome that are susceptible ("hot") and resistant ("cold") to SHM, revealing that SHM susceptibility and resistance are often properties of entire topologically associated domains (TADs). Comparison of hot and cold TADs reveals that while levels of transcription are equivalent, hot TADs are enriched for the cohesin loader NIPBL, super-enhancers, markers of paused/stalled RNA polymerase 2, and multiple important B cell transcription factors. We demonstrate that at least some hot TADs contain enhancers that possess SHM targeting activity and that insertion of a strong Ig SHM-targeting element into a cold TAD renders it hot. Our findings lead to a model for SHM susceptibility involving the cooperative action of cis-acting SHM targeting elements and the dynamic and architectural properties of TADs.
Project description:The Igh locus undergoes an amazing array of DNA rearrangements and modifications during B cell development. During early stages, the variable region gene is constructed from constituent variable (V), diversity (D), and joining (J) segments (VDJ joining). B cells that successfully express an antibody can be activated, leading to somatic hypermutation (SHM) focused on the variable region, and class switch recombination (CSR), which substitutes downstream constant region genes for the originally used Cμ constant region gene. Many investigators, ourselves included, have sought to understand how these processes specifically target the Igh locus and avoid other loci and potential deleterious consequences of malignant transformation. Our laboratory has concentrated on a complex regulatory region (RR) that is located downstream of Cα, the most 3' of the Igh constant region genes. The ~40 kb 3' RR, which is predicted to serve as a downstream major regulator of the Igh locus, contains two distinct segments: an ~28 kb region comprising four enhancers, and an adjacent ~12 kb region containing multiple CTCF and Pax5 binding sites. Analysis of targeted mutations in mice by a number of investigators has concluded that the entire 3' RR enhancer region is essential for SHM and CSR (but not for VDJ joining) and for high levels of expression of multiple isotypes. The CTCF/Pax5 binding region is a candidate for influencing VDJ joining early in B cell development and serving as a potential insulator of the Igh locus. Components of the 3' RR are subject to a variety of epigenetic changes during B cell development, i.e., DNAse I hypersensitivity, histone modifications, and DNA methylation, in association with transcription factor binding. I propose that these changes provide a foundation by which regulatory elements in modules of the 3' RR function by interacting with each other and with target sequences of the Igh locus.
Project description:The Aicda locus encodes the activation induced cytidine deaminase (AID) and is highly expressed in germinal center (GC) B cells to initiate somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes. Besides these Ig specific activities in B cells, AID has been implicated in active DNA demethylation in non-B cell systems. We here determined a potential role of AID as an epigenetic eraser and transcriptional regulator in B cells. RNA-Seq on different B cell subsets revealed that Aicda(-/-) B cells are developmentally affected. However as shown by RNA-Seq, MethylCap-Seq, and SNP analysis these transcriptome alterations may not relate to AID, but alternatively to a CBA mouse strain derived region around the targeted Aicda locus. These unexpected confounding parameters provide alternative, AID-independent interpretations on genotype-phenotype correlations previously reported in numerous studies on AID using the Aicda(-/-) mouse strain.
Project description:Ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine, facilitating DNA demethylation and generating new epigenetic marks. Here we show that concomitant loss of Tet2 and Tet3 in mice at early B cell stage blocked the pro- to pre-B cell transition in the bone marrow, decreased Irf4 expression and impaired the germline transcription and rearrangement of the Ig? locus. Tet2/3-deficient pro-B cells showed increased CpG methylation at the Ig? 3' and distal enhancers that was mimicked by depletion of E2A or PU.1, as well as a global decrease in chromatin accessibility at enhancers. Importantly, re-expression of the Tet2 catalytic domain in Tet2/3-deficient B cells resulted in demethylation of the Ig? enhancers and restored their chromatin accessibility. Our data suggest that TET proteins and lineage-specific transcription factors cooperate to influence chromatin accessibility and Ig? enhancer function by modulating the modification status of DNA.
Project description:DNA lesions inflicted by activation-induced deaminase (AID) instrumentally initiate the processes reshaping immunoglobulin genes in mature B-cells, from local somatic hypermutation (SHM) to junctions of distant breaks during class switch recombination (CSR). It remains incompletely understood how these divergent outcomes of AID attacks are differentially and temporally focused, with CSR strictly occurring in the Ig heavy chain (IgH) locus while SHM concentrates on rearranged V(D)J regions in the IgH and Ig light chain loci. In the IgH locus, disruption of either the 3'Regulatory Region (3'RR) super-enhancer or of switch (S) regions preceding constant genes, profoundly affects CSR. Reciprocally, we now examined if these elements are sufficient to induce CSR in a synthetic locus based on the Ig? locus backbone. Addition of a surrogate "core 3'RR" (c3'RR) and of a pair of transcribed and spliced Switch regions, together with a reporter system for "?-CSR" yielded a switchable Ig? locus. While the c3'RR stimulated SHM at S regions, it also lowered the local SHM threshold necessary for switch recombination to occur. The 3'RR thus both helps recruit AID to initiate DNA lesions, but then also promotes their resolution through long-distance synapses and recombination following double-strand breaks.
Project description:The 3' regulatory region (3' RR) of the murine immunoglobulin heavy chain (IgH) locus contains multiple DNase I-hypersensitive (hs) sites. Proximal sites hs3A, hs1.2, and hs3B are located in an extensive palindromic region and together with hs4 are associated with enhancers involved in the expression and class switch recombination of IgH genes. Distal hs5, -6, and -7 sites located downstream of hs4 comprise a potential insulator for the IgH locus. In pro-B cells, hs4 to -7 are associated with marks of active chromatin, while hs3A, hs1.2, and hs3B are not. Our analysis of DNA methylation-sensitive restriction sites of the 3' RR has revealed a similar modular pattern in pro-B cells; hs4 to -7 sites are unmethylated, while the palindromic region is methylated. This modular pattern of DNA methylation and histone modifications appears to be determined by at least two factors: the B-cell-specific transcription factor Pax5 and linker histone H1. In pre-B cells, a region beginning downstream of hs4 and extending into hs5 showed evidence of allele-specific demethylation associated with the expressed heavy chain allele. Palindromic enhancers become demethylated later in B-cell differentiation, in B and plasma cells.
Project description:To create high-affinity antibodies, B cells target a high rate of somatic hypermutation (SHM) to the Ig variable-region genes that encode the antigen-binding site. This mutational process requires transcription and is triggered by activation-induced cytidine deaminase (AID), which converts deoxycytidine to deoxyuridine. Mistargeting of AID to non-Ig genes is thought to result in the malignant transformation of B cells, but the mechanism responsible for targeting SHM to certain DNA regions and not to others is largely unknown. Cis-acting elements have been proposed to play a role in directing the hypermutation machinery, but the motifs required for targeting SHM have been difficult to identify because many of the candidate elements, such as promoters or enhancers, are also required for transcription of Ig genes. Here we describe a system in cultured hybridoma cells in which transcription of the endogenous heavy-chain Ig gene continues in the absence of the core intronic enhancer (Emu) and its flanking matrix attachment regions (MARs). When AID is expressed in these cells, SHM occurred at the WT frequency even when Emu and the MARs were absent together. Interestingly, SHM occurred at less than the WT frequency when Emu or the MARs were individually absent. Our results suggest that these intronic regulatory elements can exert a complex influence on SHM that is separable from their role in regulating transcription.
Project description:Immunoglobulin heavy chain (IgH) alleles have ambivalent relationships: they feature both allelic exclusion, ensuring monoallelic expression of a single immunoglobulin (Ig) allele, and frequent inter-allelic class-switch recombination (CSR) reassembling genes from both alleles. The IgH locus 3' regulatory region (3'RR) includes several transcriptional cis-enhancers promoting activation-induced cytidine deaminase (AID)-dependent somatic hypermutation (SHM) and CSR, and altogether behaves as a strong super-enhancer. It can also promote deregulated expression of translocated oncogenes during lymphomagenesis. Besides these rare, illegitimate and pathogenic interactions, we now show that under physiological conditions, the 3'RR super-enhancer supports not only legitimate cis- , but also trans-recruitment of AID, contributing to IgH inter-allelic proximity and enabling the super-enhancer on one allele to stimulate biallelic SHM and CSR. Such inter-allelic activating interactions define transvection, a phenomenon well-known in drosophila but rarely observed in mammalian cells, now appearing as a unique feature of the IgH 3'RR super-enhancer.