Individual VH promoters vary in strength, but the frequency of rearrangement of those VH genes does not correlate with promoter strength nor enhancer-independence

Love, Victoria; Lugo-Villarino, Geanncarlo; Merz, Denise; Feeney, Ann J.

doi:10.1016/s0161-5890(00)00023-7

Cited by 32 publications

(48 citation statements)

References 47 publications

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“…Germline D genes were amplified with the same upstream primers, and with a primer 3Ј of DSP, AF289 (5Ј-CCCCTAGAATTCAAGCTCCTCT TGACT). VDJ rearrangements were amplified with the same set of J H primers and with previously described V H primers (V H S107, AF72 (19); V H 7183, AF303; (18); and V H J558, AF326 (5Ј-GGGGCTTAAGCT TAGCTGTCCTGCAAGGCT)). Amplifications were performed for 35 cycles, and PCR products were cloned and sequenced as previously described (18).…”

Section: Pcr Assaymentioning

confidence: 99%

Unusual Germline DSP2 Gene Accounts for All Apparent V-D-D-J Rearrangements in Newborn, But Not Adult, MRL Mice

Kompfner

Oliveira

Montalbano

et al. 2001

The Journal of Immunology

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Anti-dsDNA autoantibodies in MRL mice contain a higher than average frequency of atypical complementarity-determining regions 3, including those made with D-D rearrangements. It has been reported that MRL mice have an intrinsically high frequency of creating VDDJ rearrangements; however, we show in this study that the majority of these apparent D-D rearrangements in B cell progenitors can be accounted for by a very novel germline DH gene in mice of the Ighj haplotype. This gene has the appearance of a D to D rearrangement due to the duplication of 9 bp common to most DSP2 genes. Germline DSP2 genes from Ighj mice were amplified, cloned, and sequenced, showing the presence of this novel gene as well as a new allele of a conventional DSP2 gene. Sequencing of D-J rearrangements revealed that Ighj mice also have a different allele of DFL16.1 and apparently lack DFL16.2. Despite the existence of this new DSP gene, analysis of VDJ rearrangements from adult bone marrow pre-B cells of MRL/lpr mice still revealed the presence of complementarity-determining region 3 containing apparent D-D joinings in 4.6% of the sequences. C3H pre-B cells had 4.2% of sequences with apparent VDDJ rearrangements, and BALB/c pre-B cells had ∼2%. DDJ intermediates were also observed, but at a lower frequency. However, strikingly, no VDDJ rearrangements were observed in newborn sequences, suggesting the process of assembly of VDJ rearrangements is fundamentally different in newborn mice vs adult mice.

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Section: Pcr Assaymentioning

confidence: 99%

Unusual Germline DSP2 Gene Accounts for All Apparent V-D-D-J Rearrangements in Newborn, But Not Adult, MRL Mice

Kompfner

Oliveira

Montalbano

et al. 2001

The Journal of Immunology

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“…The Infrequent spacer RSS binds RAG proteins 2-fold less well than an RSS with a spacer sequence that is close to consensus, and cleavage of this Infrequent RSS in cell-free assays is reduced 9-fold. Moreover, the V H S107 spacer sequence itself reduces recombination frequency in transient recombination substrate assays sufficiently to suggest that the spacer difference is a major contributing factor to the different recombination frequencies of these two V H genes in bone marrow pro-B cells (12). Hence, the spacer sequence plays a much more important role in recombination frequency than previously appreciated.…”

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confidence: 93%

V(D)J Recombination Frequencies Can Be Profoundly Affected by Changes in the Spacer Sequence

Montalbano

Ogwaro

Tang

et al. 2003

The Journal of Immunology

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Each V, D, and J gene segment is flanked by a recombination signal sequence (RSS), composed of a conserved heptamer and nonamer separated by a 12- or 23-bp spacer. Variations from consensus in the heptamer or nonamer at specific positions can dramatically affect recombination frequency, but until recently, it had been generally held that only the length of the spacer, but not its sequence, affects the efficacy of V(D)J recombination. In this study, we show several examples in which the spacer sequence can significantly affect recombination frequencies. We show that the difference in spacer sequence alone of two VHS107 genes affects recombination frequency in recombination substrates to a similar extent as the bias observed in vivo. We show that individual positions in the spacer can affect recombination frequency, and those positions can often be predicted by their frequency in a database of RSS. Importantly, we further show that a spacer sequence that has an infrequently observed nucleotide at each position is essentially unable to support recombination in an extrachromosmal substrate assay, despite being flanked by a consensus heptamer and nonamer. This infrequent spacer sequence RSS shows only a 2-fold reduction of binding of RAG proteins, but the in vitro cleavage of this RSS is ∼9-fold reduced compared with a good RSS. These data demonstrate that the spacer sequence should be considered to play an important role in the recombination efficacy of an RSS, and that the effect of the spacer occurs primarily subsequent to RAG binding.

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“…Transcription in the V H part of the locus has been detected in both the sense (same transcriptional orientation as the V H genes) and, more recently, antisense direction (3,17). The V H sense transcripts are easily seen at V H J558 genes, and sense germ-line transcripts from other V H families are present, but are far more difficult to detect (3,18). The sense transcription at V H regions begins at the V H gene promoter and ends shortly after the end of the coding region for the few transcripts that have been cloned, whereas antisense transcription is spread over intergenic regions (3,17).…”

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confidence: 99%

Noncoding transcription within the Igh distal V _H region at PAIR elements affects the 3D structure of the Igh locus in pro-B cells

Verma-Gaur

Torkamani

Schaffer

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

102

122

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Noncoding sense and antisense germ-line transcription within the Ig heavy chain locus precedes V(D)J recombination and has been proposed to be associated with Igh locus accessibility, although its precise role remains elusive. However, no global analysis of germline transcription throughout the Igh locus has been done. Therefore, we performed directional RNA-seq, demonstrating the locations and extent of both sense and antisense transcription throughout the Igh locus. Surprisingly, the majority of antisense transcripts are localized around two Pax5-activated intergenic repeat (PAIR) elements in the distal IghV region. Importantly, long-distance loops measured by chromosome conformation capture (3C) are observed between these two active PAIR promoters and Eμ, the start site of Iμ germ-line transcription, in a lineage-and stage-specific manner, even though this antisense transcription is Eμ-independent. YY1 −/− pro-B cells are greatly impaired in distal V H gene rearrangement and Igh locus compaction, and we demonstrate that YY1 deficiency greatly reduces antisense transcription and PAIR-Eμ interactions. ChIP-seq shows high level YY1 binding only at Eμ, but low levels near some antisense promoters. PAIR-Eμ interactions are not disrupted by DRB, which blocks transcription elongation without disrupting transcription factories once they are established, but the looping is reduced after heat-shock treatment, which disrupts transcription factories. We propose that transcription-mediated interactions, most likely at transcription factories, initially compact the Igh locus, bringing distal V H genes close to the DJ H rearrangement which is adjacent to Eμ. Therefore, we hypothesize that one key role of noncoding germ-line transcription is to facilitate locus compaction, allowing distal V H genes to undergo efficient rearrangement.A ntigen receptors in lymphocytes are assembled in the highly regulated lineage-specific process of V(D)J recombination, which creates a diverse repertoire of Ig and T-cell receptors. In each precursor lymphocyte, one each of the many V, D, and J gene segments at the appropriate receptor loci are juxtaposed to create a V(D)J exon encoding the variable region of the antigen receptor. In B-lineage progenitors, rearrangement occurs first at the Ig heavy chain (Igh) locus, where D H to J H rearrangement occurs first on both alleles, followed by V H to DJ H rearrangement (1). After successful rearrangement of the Igh locus, rearrangement at the Igk light chain locus begins. These successive stages of rearrangement have been proposed to be regulated by differential accessibility of different portions of the loci at the appropriate time for rearrangement (2). Early indications of this stage-specific and lineage-specific accessibility came from the observation that unrearranged gene segments underwent noncoding transcription at the stage immediately preceding their rearrangement (3, 4).The murine Igh locus spans ∼2.8 Mb, of which ∼2.4 Mb contains the 195 V H gene segments (5). The V H genes are divided i...

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Individual VH promoters vary in strength, but the frequency of rearrangement of those VH genes does not correlate with promoter strength nor enhancer-independence

Cited by 32 publications

References 47 publications

Unusual Germline DSP2 Gene Accounts for All Apparent V-D-D-J Rearrangements in Newborn, But Not Adult, MRL Mice

Unusual Germline DSP2 Gene Accounts for All Apparent V-D-D-J Rearrangements in Newborn, But Not Adult, MRL Mice

V(D)J Recombination Frequencies Can Be Profoundly Affected by Changes in the Spacer Sequence

Noncoding transcription within the Igh distal V _H region at PAIR elements affects the 3D structure of the Igh locus in pro-B cells

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Individual VH promoters vary in strength, but the frequency of rearrangement of those VH genes does not correlate with promoter strength nor enhancer-independence

Cited by 32 publications

References 47 publications

Unusual Germline DSP2 Gene Accounts for All Apparent V-D-D-J Rearrangements in Newborn, But Not Adult, MRL Mice

Unusual Germline DSP2 Gene Accounts for All Apparent V-D-D-J Rearrangements in Newborn, But Not Adult, MRL Mice

V(D)J Recombination Frequencies Can Be Profoundly Affected by Changes in the Spacer Sequence

Noncoding transcription within the Igh distal V H region at PAIR elements affects the 3D structure of the Igh locus in pro-B cells

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Noncoding transcription within the Igh distal V _H region at PAIR elements affects the 3D structure of the Igh locus in pro-B cells