Abstract:Immunoglobulin (Ig) genes naturally acquire frequent premature termination codons during the error-prone V(D)J recombination process. Although B cell differentiation is linked to the expression of productive Ig alleles, the transcriptional status of nonfunctionally recombined alleles remains unclear. Here, we tracked transcription and posttranscriptional regulation for both Ig heavy-chain (IgH) alleles in mice carrying a nonfunctional knock-in allele. We show that productively and nonproductively VDJ-rearrange… Show more
“…RNase-treated cells showed no signals, demonstrating that we were measuring RNA (Supplementary Figure S1). These results supported previously published work in which biallelic expression (19,30,31), active chromatin marks and RNA polymerase II binding (30) were found to be associated with both productive and non-productive IgH alleles in mature B cells. Figure 1.Biallelic expression and comparable nuclear positioning of the two IgH loci relative to PCH in resting and activated B cells.…”
Section: Resultssupporting
confidence: 92%
“…In 3D-FISH studies, the IgH locus was found to be peripheral also in non-B cells, whereby in EL-4 T cells both IgH alleles were associated with the nuclear lamina but not with PCH (12,44). The proposed model of mono-allelic recruitment to PCH in mature B cells (15,18) was further challenged by the observation that both productive and nonproductive IgH alleles are transcribed in activated splenic B cells (19,30). …”
In developing B cells, the immunoglobulin heavy chain (IgH) locus is thought to move from repressive to permissive chromatin compartments to facilitate its scheduled rearrangement. In mature B cells, maintenance of allelic exclusion has been proposed to involve recruitment of the non-productive IgH allele to pericentromeric heterochromatin. Here, we used an allele-specific chromosome conformation capture combined with sequencing (4C-seq) approach to unambigously follow the individual IgH alleles in mature B lymphocytes. Despite their physical and functional difference, productive and non-productive IgH alleles in B cells and unrearranged IgH alleles in T cells share many chromosomal contacts and largely reside in active chromatin. In brain, however, the locus resides in a different repressive environment. We conclude that IgH adopts a lymphoid-specific nuclear location that is, however, unrelated to maintenance of allelic exclusion. We additionally find that in mature B cells—but not in T cells—the distal VH regions of both IgH alleles position themselves away from active chromatin. This, we speculate, may help to restrict enhancer activity to the productively rearranged VH promoter element.
“…RNase-treated cells showed no signals, demonstrating that we were measuring RNA (Supplementary Figure S1). These results supported previously published work in which biallelic expression (19,30,31), active chromatin marks and RNA polymerase II binding (30) were found to be associated with both productive and non-productive IgH alleles in mature B cells. Figure 1.Biallelic expression and comparable nuclear positioning of the two IgH loci relative to PCH in resting and activated B cells.…”
Section: Resultssupporting
confidence: 92%
“…In 3D-FISH studies, the IgH locus was found to be peripheral also in non-B cells, whereby in EL-4 T cells both IgH alleles were associated with the nuclear lamina but not with PCH (12,44). The proposed model of mono-allelic recruitment to PCH in mature B cells (15,18) was further challenged by the observation that both productive and nonproductive IgH alleles are transcribed in activated splenic B cells (19,30). …”
In developing B cells, the immunoglobulin heavy chain (IgH) locus is thought to move from repressive to permissive chromatin compartments to facilitate its scheduled rearrangement. In mature B cells, maintenance of allelic exclusion has been proposed to involve recruitment of the non-productive IgH allele to pericentromeric heterochromatin. Here, we used an allele-specific chromosome conformation capture combined with sequencing (4C-seq) approach to unambigously follow the individual IgH alleles in mature B lymphocytes. Despite their physical and functional difference, productive and non-productive IgH alleles in B cells and unrearranged IgH alleles in T cells share many chromosomal contacts and largely reside in active chromatin. In brain, however, the locus resides in a different repressive environment. We conclude that IgH adopts a lymphoid-specific nuclear location that is, however, unrelated to maintenance of allelic exclusion. We additionally find that in mature B cells—but not in T cells—the distal VH regions of both IgH alleles position themselves away from active chromatin. This, we speculate, may help to restrict enhancer activity to the productively rearranged VH promoter element.
“…1D). However, because of allelic exclusion, only one of the two alleles of each IgH and Igk gene will be expressed as functional antibody chains in single cells (Vettermann and Schlissel 2010), while the transcripts arising from incompletely or nonfunctionally rearranged alleles will be selectively degraded in the cytoplasm (Tinguely et al 2012). If pairing of different Ig genes in the same transcription factories is functionally linked to optimization of antibody production, then one would predict that functionally rearranged Ig genes would be preferred partners in these structures.…”
To understand the relationships between nuclear organization and gene expression in a model system, we employed three-dimensional imaging and chromatin immunoprecipitation (ChIP)-chromosome conformation capture (3C) techniques to investigate the topographies of the immunoglobulin (Ig) genes and transcripts during B-cell development. Remarkably, in plasma cells, when antibody synthesis peaks, active Ig genes residing on three different chromosomes exhibit pronounced colocalizations in transcription factories, often near the nuclear periphery, and display trans-chromosomal enhancer interactions, and their transcripts frequently share interchromatin trafficking channels. Conceptually, these features of nuclear organization maximize coordinated transcriptional and transcript trafficking control for potentiating the optimal cytoplasmic assembly of the resulting translation products into protein multimers.
“…5,6 Third, practically all BLs carry translocations of the MYC oncogene into 1 of the Ig loci, mostly the IgH locus.…”
mentioning
confidence: 99%
“…5,6 Third, practically all BLs carry translocations of the MYC oncogene into 1 of the Ig loci, mostly the IgH locus. 7 These translocations either target 1 of the IgH switch regions or they occur as mistakes of somatic hypermutation and are then found within or close to rearranged Ig V region genes.…”
The article by Lombardo and colleagues 1 described high throughput sequencing of the immunoglobulin (Ig) heavy and light chain genes from African Burkitt lymphomas (BLs). On the basis of their analysis and, in particular, because of the frequent lack of detection of Ig D H J H or V H D H J H rearrangements on the nonexpressed Ig heavy (IgH) chain allele, as well as ongoing mutation on the nonexpressed allele, the authors conclude that BLs derive from an abnormal B-cell progenitor.I would like to point out several features of the diversification processes of human Ig genes and the pathogenesis of BLs that were not considered in the interpretation of the data and that bring into question the conclusions in the article by Lombardo et al. 3,4 The nonproductive V H D H J H rearrangements are apparently well transcribed (which is considered an essential prerequisite for being targeted by somatic hypermutation), but because of premature stop codons, the messenger RNA (mRNA) is fast degraded by the process of nonsense-mediated decay and other mechanisms, so that in the steady state, one finds few transcripts from the nonproductive allele. 5,6 Third, practically all BLs carry translocations of the MYC oncogene into 1 of the Ig loci, mostly the IgH locus.7 These translocations either target 1 of the IgH switch regions or they occur as mistakes of somatic hypermutation and are then found within or close to rearranged Ig V region genes. 4,7 In endemic BL, the MYC translocations seem to be frequent in or near rearranged IgH V region genes.8 If the translocation disrupts the nonexpressed V H D H J H rearrangements (the translocations do not disrupt the productive allele, because BLs are nearly always surface Ig-positive), then the original rearrangement cannot be further amplified by polymerase chain reaction (PCR).Fourth, it is to be expected that some V H D H J H rearrangements were not successfully amplified because of a technical failure of the PCR. The rearrangements were amplified with primers binding to the V H leader region and to the IGHJ gene segments. In both regions, somatic hypermutation occurs, and a single mutation in the Ig V region gene close to the 39 of the primer binding site will impair efficient amplification of such rearrangements. Thus, when studying mutated B cells, a fraction of mutated Ig V gene rearrangements will likely not be amplified with a strategy using primers in the somatic mutation target sequence. Moreover, about 40% of mutated nonproductive V H D H J H rearrangements of normal human B cells carry deletions and/or insertions/duplications, which are sometimes several hundred bases long and thus can also prevent successful amplification by PCR. 4 The chance to amplify mutated Ig V genes is higher when using mRNA and C-region-specific primers instead of J gene segment primers because there are no mutations in the C-region exons. But for nonproductive alleles, as pointed out above, one will likely miss them because of the low steady state mRNA levels of such rearrangements.
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