Unrearranged Allele Research Articles

A linear-amplification VDJ-seq technique for quantification of immunoglobulin and T cell receptor diversity.

The V(D)J recombination is essential for generating a highly diverse repertoire of antigen receptors expressed on T and B lymphocytes. Here, we developed a linear-amplification VDJ-seq technique for quantifying V(D)J recombination of antigen receptor genes. This technique takes advantage of linear amplification using in vitro transcription and reverse transcription to avoid bias generated by the PCR amplification of low copy number of target DNA. The unrearranged alleles are removed by in vitro cleavage with the CRISPR-Cas9 system. The linear-amplification VDJ-seq assay was applied in quantification of the Vκ-Jκ recombination of the mouse Igκ gene with Jκ capture primers. The Jκ genes were detected in 95.86% of clean reads with more than half containing the Vκ gene, indicating high specificity of capturing and amplification. We also applied this approach to quantify the usage of Jα within the Trav12 gene family of the Tcra gene.

Localized DNA Demethylation at Recombination Intermediates during Immunoglobulin Heavy Chain Gene Assembly

Author SummaryDNA methylation at CpG dinucleotides is implicated in the regulation of gene expression in mammals. However, the regulation of DNA methylation itself is less clear despite recent advances in identifying enzymes that add or remove methyl groups. We have investigated the dynamics of DNA methylation during genome rearrangements that assemble antigen receptor genes in developing B lymphocytes to determine whether methylation status correlates with rearrangement potential. Two recombination events generate immunoglobulin heavy chain (IgH) genes. The first step brings together diversity (DH) and joining (JH) gene segments to produce DJH junctions. We show that both gene segments are methylated prior to rearrangement, whereas the DJH product is demethylated. DJH junctional demethylation is tissue-specific and requires an enhancer, Eμ, located within the IgH locus. The latter observations indicate that localized demethylation of the DJH junction occurs after the first recombination step and thus does not guide this first step of IgH gene assembly. Our working hypothesis is that recombination induces demethylation of recombinant product and may mark the junction for the second step of IgH rearrangement, juxtaposition of variable (VH) gene segments to rearranged DJH products to produce fully recombined V(D)J alleles.

Differential Regulation of Proximal and Distal Vβ Segments Upstream of a Functional VDJβ1 Rearrangement upon β-Selection

Developmental stage-specific regulation of transcriptional accessibility helps control V(D)J recombination. Vβ segments on unrearranged TCRβ alleles are accessible in CD4(-)/CD8(-) (double-negative [DN]) thymocytes, when they recombine, and inaccessible in CD4(+)/CD8(+) (double-positive [DP]) thymocytes, when they do not rearrange. Downregulation of Vβ accessibility on unrearranged alleles is linked with Lat-dependent β-selection signals that inhibit Vβ rearrangement, stimulate Ccnd3-driven proliferation, and promote DN-to-DP differentiation. Transcription and recombination of Vβs on VDJβ-rearranged alleles in DN cells has not been studied; Vβs upstream of functional VDJβ rearrangements have been found to remain accessible, yet not recombine, in DP cells. To elucidate contributions of β-selection signals in regulating Vβ transcription and recombination on VDJβ-rearranged alleles, we analyzed wild-type, Ccnd3(-/-), and Lat(-/-) mice containing a preassembled functional Vβ1DJCβ1 (Vβ1(NT)) gene. Vβ10 segments located just upstream of this VDJCβ1 gene were the predominant germline Vβs that rearranged in Vβ1(NT/NT) and Vβ1(NT/NT)Ccnd3(-/-) thymocytes, whereas Vβ4 and Vβ16 segments located further upstream rearranged at similar levels as Vβ10 in Vβ1(NT/NT)Lat(-/-) DN cells. We previously showed that Vβ4 and Vβ16, but not Vβ10, are transcribed on Vβ1(NT) alleles in DP thymocytes; we now demonstrate that Vβ4, Vβ16, and Vβ10 are transcribed at similar levels in Vβ1(NT/NT)Lat(-/-) DN cells. These observations indicate that suppression of Vβ rearrangements is not dependent on Ccnd3-driven proliferation, and DN residence can influence the repertoire of Vβs that recombine on alleles containing an assembled VDJCβ1 gene. Our findings also reveal that β-selection can differentially silence rearrangement of germline Vβ segments located proximal and distal to functional VDJβ genes.

Repeat Organization and Epigenetic Regulation of the DH-Cμ Domain of the Immunoglobulin Heavy-Chain Gene Locus

The first steps of murine immunoglobulin heavy-chain (IgH) gene recombination take place within a chromosomal domain that contains diversity (D(H)) and joining (J(H)) gene segments, but not variable (V(H)) gene segments. Here we show that the chromatin state of this domain is markedly heterogeneous. Specifically, only 5'- and 3'-most D(H) gene segments carry active chromatin modifications, whereas intervening D(H)s are associated with heterochromatic marks that are maintained by ongoing histone deacetylation. The intervening D(H)s form part of a tandemly repeated sequence that expresses tissue-specific, antisense oriented transcripts. We propose that the intervening D(H) genes are actively suppressed by repeat-induced epigenetic silencing, which is reflected in their infrequent representation in DJ(H) junctions compared to the flanking D(H) genes.

Exclusion is spreading

Allelic exclusion simplifies the problem of immune recognition. It ensures that each B cell makes antibodies that recognize only one target because, once the DNA of a single immunoglobulin allele has rearranged to form a functional gene, the other allele is prevented from rearranging. Now Esther Roldan, Jane Skok (University College London, UK), and colleagues find that allele exclusion may rely on putting different parts of the unrearranged gene too far from each other to allow rearrangement. Rearrangement is only possible in such huge loci because of DNA contraction and (as shown here) looping that juxtaposes distant DNA sites. Skok and colleagues now show that successful rearrangement is immediately followed by decontraction of the unrearranged allele. Proximal variable (V) regions are still within reach of the rearrangement apparatus, but the products of these rearrangements are disfavored later because they pair poorly with immunoglobulin light chains and usually fail to induce positive selection of cells. There is a backup mechanism for allele exclusion. Coincident with decontraction, the unrearranged allele is also recruited to repressive centromeric domains. But Skok says she thinks decontraction “is the most important factor. Decontraction seems to be irreversible and immediate, whereas recruitment is reversible.” Reference: Roldan, E., et al. 2004. Nat. Immunol. doi:.10.1038/ni1150 [PubMed] [Cross Ref]

Selection for B cells with productive IgL gene rearrangements occurs in the bursa of Fabricius during chicken embryonic development.

The vast majority of immunoglobulin-expressing mature chicken B lymphocytes contain one functionally rearranged and one unrearranged allele of the immunoglobulin light chain (IgL) gene. Therefore, nearly all IgL V-J rearrangements present in mature chickens are in-frame. In contrast, the Ig genes of mature mammalian B cells contain a high proportion of out-of-frame V-J joints. To investigate the basis for this difference, gene rearrangement at the chicken IgL locus was characterized during embryonic development and in mature B-cell lines. Joining of the single functional variable (VL) segment with the single joining (JL) segment occurs in cells in multiple tissues during a transient period of chicken embryogenesis. Only one-third of the V-J joints cloned from days 10-12 of development are in-frame. An increasing proportion of in-frame V-J joints is observed within the bursa of Fabricius at successively later stages of development. Our data suggest that the bursa of Fabricius serves during embryonic development as a site of selective amplification of cells that have undergone productive V-J joining, such that nearly all V-J joints present in postembryonic B cells are in-frame. The high frequency of rearranged alleles joined in-frame that is found in posthatching bursal cells and mature B-cell lines appears to result from a low frequency with which cells undergo IgL rearrangement at both alleles, rather than from an increase in the precision of V-J joining in avian species.

Disruption of the putative c-myc auto-regulation mechanism in a human B cell line.

It has been proposed that excessive or temporally inappropriate expression of the c-myc proto-oncogene determines a genetic program for cells that directs them toward proliferation, and away from differentiation. In support of this proposal, constitutive expression of c-myc in either murine erythroleukaemia cells, or 3T3-L1 cells largely prevents the progression of their respective differentiation schedules(1,2). Additionally, when resting cells of numerous types are treated with various mitogens, proliferation is preceded by rapid induction of c-myc expression (3,4). The inductive role that the mitogens play in proliferation can be in part substituted for by c-myc (5). An issue that remains outstanding is how the induction of c-myc is controlled. It has been theorised that c-myc auto-regulates its own transcription (6). This was provoked by the observation that in Burkitts lymphoma (BL), there is de-regulated expression of c-myc derived from the translocated allele, whilst the unrearranged allele is generally silent (6). This situation was to some extent re-capitulated by Lombardi et al (7) who witnessed the down-regulation of c-myc genes in human B cell lines into which they had stably introduced de-regulated c-myc. Because the products of the c-myc gene are two nuclear phosphoproteins with DNA-binding capacity, it has been considered that the auto-regulation is direct, requiring only the c-myc product and the c-myc template. Alternatively, the effect may be indirect, operating via pathways that may be disrupted and hence rendered inoperative in certain cell types. In order to examine this, we have investigated the generality of auto-regulation. Here we report an established human B cell line that is not susceptible to auto-regulation.KeywordsBurkitts LymphomaBurkitts LymphomaNuclear PhosphoproteinCell BioIMetallothionein PromoterThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Insertion of an Alu SINE in the human homologue of the Mlvi-2 locus.

Fifty-nine human DNA samples derived from either normal tissues or hematopoietic neoplasias were examined for rearrangements in the Mlvi-2 locus, a putative oncogene. The rearranged Mlvi-2 sequences in one of them, a B cell lymphoma, were shown to result from the insertion of an approximately 300 bp DNA fragment that hybridized to a human Alu probe. DNA sequence analysis of both the rearranged and the nonrearranged allele around the site of the insertion revealed the following: a) the insert was 88.4% homologous to the consensus sequence of the Alu family of repeats and 75% homologous to the Alu related sequence in the human 7SL RNA; b) similar to other sequenced SINES, a poly(d.A) tract was present at the 3' end of this element; c) an 8 bp direct repeat was present at both ends of the inserted element; d) this repeat was present as a single copy in the unrearranged allele. We conclude from these findings that: Alu sequences can transpose and that the direct repeats flanking certain Alu SINES may be generated by the duplication of single copy cellular sequences at the site of the insertion. Furthermore the recent nature of the Alu insertion in the Mlvi-2 locus coupled to the low degree of homology of the inserted Alu to the Alu related sequence in the 7SL RNA suggest that this event did not occur via reverse transcription and reintegration of the 7SL RNA.