Complete Sequence Assembly and Characterization of the C57BL/6 Mouse Ig Heavy Chain V Region

Johnston, C L; Wood, Andrew L.; Bolland, Daniel J.; Corcoran, Anne E.

doi:10.4049/jimmunol.176.7.4221

Cited by 141 publications

(174 citation statements)

References 96 publications

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“…S1), and the DNA derived from the mononucleosome fraction arising from each (50). The IgH 3′ regulatory region (3′RR, green), Eμ enhancer (Eμ, green), CTCF binding elements (CBE1/2, red) (70,71), and PAX5-dependent regulatory (PAIR) elements (43) are indicated below the locus.…”

Section: Resultsmentioning

confidence: 99%

Regulated large-scale nucleosome density patterns and precise nucleosome positioning correlate with V(D)J recombination

Pulivarthy

Lion

Kuzu

et al. 2016

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

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We show that the physical distribution of nucleosomes at antigen receptor loci is subject to regulated cell type-specific and lineagespecific positioning and correlates with the accessibility of these gene segments to recombination. At the Ig heavy chain locus (IgH), a nucleosome in pro-B cells is generally positioned over each IgH variable (VH) coding segment, directly adjacent to the recombination signal sequence (RSS), placing the RSS in a position accessible to the recombination activating gene (RAG) recombinase. These changes result in establishment of a specific chromatin organization at the RSS that facilitates accessibility of the genomic DNA for the RAG recombinase. In contrast, in mouse embryonic fibroblasts the coding segment is depleted of nucleosomes, which instead cover the RSS, thereby rendering it inaccessible. Pro-T cells exhibit a pattern intermediate between pro-B cells and mouse embryonic fibroblasts. We also find large-scale variations of nucleosome density over hundreds of kilobases, delineating chromosomal domains within IgH, in a cell typedependent manner. These findings suggest that developmentally regulated changes in nucleosome location and occupancy, in addition to the known chromatin modifications, play a fundamental role in regulating V(D)J recombination. Nucleosome positioning-which has previously been observed to vary locally at individual enhancers and promoters-may be a more general mechanism by which cells can regulate the accessibility of the genome during development, at scales ranging from several hundred base pairs to many kilobases.T he diverse repertoire of antigen receptors in vertebrates is generated by an ordered series of somatic site-specific DNA rearrangement events, collectively termed V(D)J recombination. Antigen receptor genes are assembled from V, D, and J gene segments organized into seven different loci [T-cell receptor (TCR) α, β, γ, and δ, and Ig H, κ, and λ], each of which undergoes a series of recombination reactions to generate a functional TCR or B-cell receptor (BCR) (1, 2) Rearrangement is lineage-specific, such that BCR genes are fully rearranged only in B cells and TCR genes are assembled only in T cells. Rearrangement also occurs in a preferred temporal order. For example, at the human and murine IgH loci, joining of D to J segments precedes V to DJ joining, and IgH joining precedes joining at Igκ (or Igλ) (3). In addition, recombination at several loci shows "allelic exclusion": Functional VDJH or VJκ/Jλ joining occurs only on one allele (4).Recombination is initiated by the lymphocyte-specific recombination activating gene (RAG)1/RAG2 endonuclease. RAG1/2 cleaves at the recombination signal sequences (RSSs) flanking all antigen receptor gene segments. The consensus RSS consists of a heptamer sequence directly adjacent to the coding element and an A/T-rich nonamer separated from the heptamer by a spacer region of conserved length (12 or 23 bp) but relatively nonconserved sequence. The broken ends are then joined with the aid of DNA repair proteins,...

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Section: Resultsmentioning

confidence: 99%

Regulated large-scale nucleosome density patterns and precise nucleosome positioning correlate with V(D)J recombination

Pulivarthy

Lion

Kuzu

et al. 2016

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

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“…These bacterial artificial chromosomes provide at least 2-fold coverage except in four regions of 12,735, 1087, 528, and 430 bp, which have single strand coverage. Sequence analysis was performed using Nucleotide Identity X, provided by the Human Genome Mapping Project (21) Real-time and Strand-specific RT-PCR-RNA was purified using the RNeasy kit (Qiagen). DNA was removed using the RQ1 DNase I kit (Promega).…”

Section: Mice Cell Lines and Cell Sorting-rag1mentioning

confidence: 99%

“…We have previously assembled the V and D region sequences of the C57BL/6 mouse Igh locus (10,21), revealing that they are separated by 96 kb of DNA sequence. Here we test the hypothesis that this uncharacterized region contains cis-acting regulatory elements, strategically positioned to influence ordered V(D)J recombination.…”

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confidence: 99%

The Mouse Immunoglobulin Heavy Chain V-D Intergenic Sequence Contains Insulators That May Regulate Ordered V(D)J Recombination

Featherstone¹,

Wood²,

Bowen³

et al. 2010

Journal of Biological Chemistry

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V(D)J recombination of the multigene antigen receptor loci is essential for the generation of a diverse antigen receptor repertoire. Recombination is strictly regulated, occurring only in lymphocytes due to restricted expression of the recombination activating gene enzymes, RAG1 and RAG2, therein. Further, T cell receptors only recombine in T cells, B cell receptors only recombine in B cells, and the loci only recombine at specific stages in lymphocyte differentiation. In B cells, the Igh recombines before the Ig light chains. Finally some antigen receptor loci (e.g. the Igh) have two ordered recombination events. A D gene first recombines with a J gene on both alleles, followed by recombination of a V gene to the DJ recombined segment. Once a productive VDJ rearrangement has been generated, further V to DJ recombination is prevented on the second allele, a process termed allelic exclusion, which in B cells ensures that each B cell expresses a monoclonal IgH (1).Ordered recombination is crucial for antigen receptor integrity, but key questions remain: how is recombination order achieved, and how is it regulated? Numerous studies have suggested that order is achieved through alterations in the chromatin conformation of individual gene domains at sequential stages of lymphocyte development (2). In the mouse Igh locus, the D-J-C region acquires histone post-translational modifications characteristic of open chromatin before the V region (3, 4). Non-coding RNA transcripts, including I, generated from E, located 3Ј of the J genes (5), and o, transcribed from the promoter of the most 3Ј D gene, DQ52, occur on germ line alleles (6). Following D to J recombination, non-coding transcripts are generated from the V genes (7,8). Furthermore, extensive antisense intergenic transcription occurs throughout the D and J domains before D to J, and then throughout the V domain before V to DJ recombination (9, 10). Nuclear positioning may also play a role in ordered V(D)J recombination. The Igh locus is tethered at the nuclear periphery via the V region in non-B cells (11,12). Relocation toward euchromatic regions occurs preferentially from the DJC end, favoring D to J recombination. Furthermore, locus compaction through DNA looping is required for distal V gene recombination (13,14). Several transcription factors, including Pax5 (13), YY1 (15), and Ikaros (16), play a role in looping, and in their absence, only the D-proximal V genes recombine. Following productive V(D)J recombination and cell surface expression of an IgH polypeptide, several of the above processes are reversed to silence V to DJ recombination of the second allele by allelic exclusion. Both Igh V regions decontract, V region germ line transcription is lost, and the second Igh allele is recruited to pericentric heterochromatin via the D-distal V genes (1). In contrast, both DJC regions remain transcriptionally active (9, 17). Thus, there is differential chromatin regulation of both activation and inactivation of the DJC versus V regions of the Igh locus.

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“…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 into 16 V H gene families based on sequence homology.…”

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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.

101

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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Complete Sequence Assembly and Characterization of the C57BL/6 Mouse Ig Heavy Chain V Region

Cited by 141 publications

References 96 publications

Regulated large-scale nucleosome density patterns and precise nucleosome positioning correlate with V(D)J recombination

Regulated large-scale nucleosome density patterns and precise nucleosome positioning correlate with V(D)J recombination

The Mouse Immunoglobulin Heavy Chain V-D Intergenic Sequence Contains Insulators That May Regulate Ordered V(D)J Recombination

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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Complete Sequence Assembly and Characterization of the C57BL/6 Mouse Ig Heavy Chain V Region

Cited by 141 publications

References 96 publications

Regulated large-scale nucleosome density patterns and precise nucleosome positioning correlate with V(D)J recombination

Regulated large-scale nucleosome density patterns and precise nucleosome positioning correlate with V(D)J recombination

The Mouse Immunoglobulin Heavy Chain V-D Intergenic Sequence Contains Insulators That May Regulate Ordered V(D)J Recombination

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