Cryptic Recombination Signal Sequences Research Articles

Molecular characterization of TCF3::PBX1 chromosomal breakpoints in acute lymphoblastic leukemia and their use for measurable residual disease assessment

The translocation t(1;19)(q23;p13) with the resulting chimeric TCF3::PBX1 gene is the third most prevalent recurrent chromosomal translocation in acute lymphoblastic leukemia and accounts for 3–5% of cases. The molecular background of this translocation has been incompletely studied, especially in adult cases. We characterized the chromosomal breakpoints of 49 patients with TCF3::PBX1 and the corresponding reciprocal PBX1::TCF3 breakpoints in 15 cases at the molecular level, thus providing an extensive molecular overview of this translocation in a well-defined study patient population. Breakpoints were found to be remarkably clustered not only in TCF3 but also in PBX1. No association with DNA repeats or putative cryptic recombination signal sequence sites was observed. A simplified detection method for breakpoint identification was developed and the feasibility of patient-specific chromosomal break sites as molecular markers for detecting measurable residual disease (MRD) was explored. A highly sensitive generic real-time PCR for MRD assessment using these breakpoint sequences was established that could serve as a useful alternative to the classical method utilizing rearranged immune gene loci. This study provides the first extensive molecular data set on the chromosomal breakpoints of the t(1;19)/TCF3::PBX1 aberration in adult ALL. Based on the obtained data a generic MRD method was developed that has several theoretical advantages, including an on average higher sensitivity and a greater stability of the molecular marker in the course of disease.

Nuclear corepressors NCOR1/NCOR2 regulate B cell development, maintain genomic integrity and prevent transformation.

The nuclear corepressors NCOR1 and NCOR2 interact with transcription factors involved in B cell development and potentially link these factors to alterations in chromatin structure and gene expression. Herein, we demonstrate that Ncor1/2 deletion limits B cell differentiation via impaired recombination, attenuates pre-BCR signaling and enhances STAT5-dependent transcription. Furthermore, NCOR1/2-deficient B cells exhibited derepression of EZH2-repressed gene modules, including the p53 pathway. These alterations resulted in aberrant Rag1 and Rag2 expression and accessibility. Whole-genome sequencing of Ncor1/2 DKO B cells identified increased number of structural variants with cryptic recombination signal sequences. Finally, deletion of Ncor1 alleles in mice facilitated leukemic transformation, whereas human leukemias with less NCOR1 correlated with worse survival. NCOR1/2 mutations in human leukemia correlated with increased RAG expression and number of structural variants. These studies illuminate how the corepressors NCOR1/2 regulate B cell differentiation and provide insights into how NCOR1/2 mutations may promote B cell transformation.

High‐Throughput Characterization of VDJ Recombination Signal Sequences

Developing B- and T-cells produce a diverse antigen receptor repertoire by rearranging antigen receptor genes during VDJ recombination. Recombination activating proteins, RAG1 and RAG2, catalyze VDJ recombination by cleaving DNA between recombination signal sequences (RSS) and antigen receptor gene segments, and the double-strand breaks are repaired by nonhomologous end-joining machinery. The RSS is defined as containing a consensus heptamer and a nonamer sequence separated by a less conserved spacer sequence. However, many RSSs deviate from the consensus sequence. Therefore, RAG1/2 must be promiscuous to facilitate recombination of poorly conserved RSSs, but it must also be precise to avoid off-target cryptic RSSs. Recent cryo-EM and X-ray crystallographic studies on RAG1/2 showed the RSS heptamer undergoes a dramatic structural transition in the RAG1/2 active site. Before cleavage, the heptamer region untwists by 180°, so RAG1/2 sidechains contacting the major groove switch to contacting the minor groove and vice-versa. RAG1/2 also makes few base-specific contacts prior to nicking. Therefore, we hypothesized that sequence-specific structural properties of the RSS heptamer plays a role in RAG1/2 specificity by facilitating these structural transitions in the RAG1/2 active site. To test this hypothesis and better characterize the DNA sequence specificity of RAG1/2, we modified an episomal-based VDJ recombination assay to quantify RAG1/2 activity in an unbiased approach using a large sequence-diverse set of RSSs. While the consensus heptamer sequence was highly preferred, RAG1/2 showed similarly high activity on many different sequences. Notably, purine/pyrimidine (R/Y) content of RSS heptamers was a strong predictor of RAG1/2 activity, preferring alternating R/Y sequence motifs at nucleotide positions 5-7 of the RSS heptamer. RAG1/2 also favored A+T base-pairs in the first two positions of the RSS spacer. These data show RAG1/2 is highly active on many DNA sequences deviating from the consensus RSS, and R/Y content of RSS heptamers is a good predictor of RAG activity. Molecular dynamics simulations of RSSs using the parmbsc1 force field showed that DNA sequences preferred by RAG1/2 had unique twist distributions, particularly between nucleotide positions 5-7. In conclusion, these data support our hypothesis that RAG1/2 can recognize many different DNA sequences, and sequence-specific structural features of alternating R/Y nucleotides promote RAG1/2 cleavage. This mechanism of promiscuously selecting sequences for VDJ recombination yields a diverse antigen receptor repertoire, but it also highlights the constant threat of off-target VDJ recombination events. Further investigation of RAG1/2 specificity will help elucidate the genetic instructions guiding VDJ recombination to antigen receptor gene segments.

IKZF1 Deletions with COBL Breakpoints Are Not Driven by RAG-Mediated Recombination Events in Acute Lymphoblastic Leukemia

IKZF1 deletion (ΔIKZF1) is an important predictor of relapse in both childhood and adult B-cell precursor acute lymphoblastic leukemia (B-ALL). Previously, we revealed that COBL is a hotspot for breakpoints in leukemia and could promote IKZF1 deletions. Through an international collaboration, we provide a detailed genetic and clinical picture of B-ALL with COBL rearrangements (COBL-r). Patients with B-ALL and IKZF1 deletion (n = 133) were included. IKZF1 ∆1-8 were associated with large alterations within chromosome 7: monosomy 7 (18%), isochromosome 7q (10%), 7p loss (19%), and interstitial deletions (53%). The latter included COBL-r, which were found in 12% of the IKZF1 ∆1-8 cohort. Patients with COBL-r are mostly classified as intermediate cytogenetic risk and frequently harbor ETV6, PAX5, CDKN2A/B deletions. Overall, 56% of breakpoints were located within COBL intron 5. Cryptic recombination signal sequence motifs were broadly distributed within the sequence of COBL, and no enrichment for the breakpoint cluster region was found. In summary, a diverse spectrum of alterations characterizes ΔIKZF1 and they also include deletion breakpoints within COBL. We confirmed that COBL is a hotspot associated with ΔIKZF1, but these rearrangements are not driven by RAG-mediated recombination.

Abstract B171: Elucidating the mechanism of RAG tracking and its impacts on V(D)J recombination

Abstract RAG endonuclease initiates V(D)J recombination by binding to the recombination signal sequences (RSSs), introducing DNA breaks between two V(D)J gene segments, and mediating their joining. This process is tightly regulated to generate diverse antigen receptor repertoires and prevent aberrant events that could cause genomic translocations/deletions and lymphoid cancer. RAG can track directionally over chromosomal loop domain in which they reside. Tracking is evidenced by cleavage/joining of short cryptic RSSs lying exclusively in convergent orientation to a bona fide RSS. However, the mechanism of RAG tracking and its impacts remain unknown. Here I established an unbiased assay system by utilizing allele-specific barcode and employing high throughput genome-wide translocation sequencing (HTGTS) to quantitatively evaluate RAG-mediated recombination frequency. I found that insertion of one single bona fide RSS is sufficient to initiate RAG tracking in the c-Myc domain and the recombination between the RSS and its cryptic RSSs seems to follow the 12/23 rule to some degree. When ectopically inserted bona fide RSS pair is 0.5kb apart, convergent configuration of the RSS pair is 4.8-fold more frequent than the tandem configuration. When the distance increases to 267kb, the difference increases to 14-fold, suggesting the contribution of RAG tracking over diffusion-based mechanism increases when the distance between RSS pair increases. In addition, the presence of CTCF-binding element promotes recombination by more than 10-fold. Depletion of Wapl, cohesin remover, to disrupt cohesin turnover reduces V(D)J recombination. The distal RSSs are much preferentially affected. The data suggest that RAG tracking may be driven by cohesin sliding along chromatin. Citation Format: Cheng-Sheng Lee, Jiazhi Hu, Frederick W. Alt. Elucidating the mechanism of RAG tracking and its impacts on V(D)J recombination [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B171.

Development of a Fluorescence in Situ Hybridization Probe for Detecting IKZF1 Deletion Mutations in Patients with Acute Lymphoblastic Leukemia

Intragenic deletion of IKZF1 is a recurrent genomic alteration in acute lymphoblastic leukemia. The deletions are mediated by illegitimate variable(diversity)joining recombination via cryptic recombination signal sequences (RSSs). We developed a fluorescence in situ hybridization (FISH) probe set that can detect any type of IKZF1 deletion, including the commonly deleted exon 4 to 7 region. The probe set consists of a designed probe for the commonly deleted region (Cy3; red) and a bacterial artificial chromosomes clone probe for detecting the 3' flanking region (Spectrum Green). Intact IKZF1 showed a fusion signal, and the deleted allele showed loss of the red signal (0R1G1F). The FISH probes worked correctly for human leukemic cell lines and clinical samples. One case showed an atypical break-apart signal (1R1G1F). Inverse PCR of the case revealed rearrangement of the excised IKZF1 fragment into a legitimate RSS site at Ig κ on chromosome 2, suggesting a pathogenic role of this recombination-activating gene 1/2-mediated event. In this study, we established FISH probe detecting IKZF1 deletion in a quick, quantitative, and cost-effective manner, and the results provided a novel insight into B-cell receptor editing by rearrangement of a cryptic RSS-mediated genomic fragment in acute lymphoblastic leukemia pathology.

Extensive RAG-Mediated Rearrangements and Mutations in BCR-ABL1 and BCR-ABL1-like Adult Acute Lymphoblastic Leukemia

BCR-ABL1 and BCR-ABL1-like acute lymphoblastic leukemia (ALL) are two major pre-B cell acute leukemia subtypes characterized by genetic alterations affecting lymphoid-specific transcription factors. Studies examining the chain of genetic events necessary to develop leukemia established that the BCR-ABL1 fusion gene and kinase-activating BCR-ABL1-like lesions are initiating events, however, insufficient for leukemia development. Secondary genetic events targeting B cell development genes are therefore an essential requirement for overt ALL. A recent study (Papaemmanuil et al, Nat. Genet., 2014) revealed that illegitimate RAG-mediated recombination is the predominant mutational mechanism establishing these secondary genetic events in ETV6-RUNX1 ALL. Of note, ETV6-RUNX1ALL is mainly restricted to pediatric cases and it remains unanswered whether this mutational process also plays a prominent role in adult ALL pathogenesis.We carried out a detailed genomic characterization to determine whether aberrant RAG activity is also a prominent mutational driver in certain adult B cell ALL (B-ALL) subtypes. Diagnostic material of 53 unselected B-ALL cases and matched remission specimens were characterized using DNA mapping arrays to discern copy number alterations (CNAs). We observed multiple BCR-ABL1/BCR-ABL1-like patients with abundant genetic lesions and selected 5 cases for targeted sequencing of CNA boundaries to determine whether these lesions were driven by RAG-mediated recombination. Whole genome sequencing (WGS) for a single BCR-ABL1-like patient was used to asses this mutational mechanism genome-wide.In total 64 structural variants (SVs) could be analyzed at base-pair level. De novo motif detection on breakpoint sequences revealed the prominence of the heptamer CACAGTG (E-value=5.68x10-91), a constituent of the recombination signal sequence (RSS), present in 121 out of 128 breakpoints (94.5%). RSS detection revealed that 58 out of 64 SVs (90.6%) had a cryptic RSS (cRSS) on one or both sides of the lesion. Incorporation of non-templated sequences was observed for 54 out of the 64 (84.4%) SVs. Superimposition of breakpoints on chromatin marks revealed a strong enrichment for active promoters and enhancers (p < 2.2x10-16). WGS data revealed cRSS motifs and incorporation of non-templated sequences for 23 out of 26 SVs (88.5%).Integrative analysis of all 6 cases confirmed 125 unique SV breakpoints strongly enriched for the active chromatin marks H3K4me3 and H3K27ac. STAT5 binding, a postulated regulator of V(D)J recombination, is similarly enriched at the breakpoints. Promiscuous binding of RAG1 and RAG2 was previously noted in human thymocytes and murine pre-B cells (Teng et al, Cell, 2015). Strikingly, the breakpoints are frequently bound by RAG2 in human thymocytes. In total 66 out of 125 breakpoints could be translated to the murine genome and revealed a strong enrichment of RAG1 and RAG2 binding at homologous positions in murine pre-B cells.Exhaustive mutation detection revealed complex somatic mutations within cRSS motifs, which are rare V(D)J recombination products introduced by erroneous cleavage and error-prone repair (open-and-shut joints). Strikingly, 4 out of 6 BCR-ABL1/BCR-ABL1-like cases had mutations in the BTLA promoter-situated cRSS, frequently in combination with a RAG-mediated deletion of the other allele (Figure 1). Genomic screening in 142 B-ALL patients confirmed 8 additional cases with BTLA promoter mutations, predominantly (6 out of 8) belonging to the BCR-ABL1/BCR-ABL1-like subgroups.We provide strong evidence that aberrant RAG activity plays a pivotal role in the development of BCR-ABL1/BCR-ABL1-like adult ALL. We demonstrate that breakpoints are strongly enriched for RAG binding implying a predisposition for illegitimate V(D)J recombination. Importantly, we report on a novel mutational mechanism introducing mutations in cRSS motifs through open-and-shut joints, frequently resulting in the biallelic inactivation of BTLA. Proliferation and V(D)J recombination during pre-B cell development is orchestrated by the interplay of IL7R and pre-BCR signalling. Strikingly, most kinase-activating lesions constitutively activate these signalling cascades and could enact, in concert with BTLA inactivation, constant proliferation, pro-survival and V(D)J recombination-initiating signals with disastrous consequences. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Illegitimate RAG-mediated recombination events are involved in IKZF1 Δ3-6 deletion in BCR-ABL1 lymphoblastic leukaemia.

Breakpoint cluster region-Abelson murine leukaemia viral oncogene homologue 1 (BCR-ABL1), encoded by the Philadelphia (Ph) chromosome, is the characteristic of chronic myeloid leukaemia (CML) and a subset of acute lymphoblastic leukaemia (ALL). We demonstrated that expression of the Ik6 transcript, which lacked exons 3-6, was observed exclusively in BCR-ABL1(+) B ALL and lymphoid blast crisis CML (BC-CML) patients harbouring the IKZF1 Δ3-6 deletion. To confirm the hypothesis that illegitimate recombination activating gene protein (RAG)-mediated recombination events are involved in IKZF1 Δ3-6 deletion in BCR-ABL1 lymphoblastic leukaemia, we first demonstrated that the expression rates of RAG1 and RAG2, collectively called RAG, were higher in ALL and BC-CML (lymphoid). Notably, analysis of relationships among RAG, BCR-ABL1 and Ikaros 6 (Ik6) showed that Ik6 can be generated only if RAG and BCR-ABL1 are co-existing. The sequencing data showed that the deleted segments of introns 2 and 6 contained cryptic recombination signal sequences (cRSSs) and frequently had non-template nucleotides inserted between breakpoints. Furthermore, we used chromatin immunoprecipitation (ChIP) technology and demonstrated that the sequences directly flanking IKZF1 Δ3-6 deletion breakpoints have significantly higher levels of histone H3 lysine 4 trimethylation (H3K4me3) modifications. Overall, RAG expression, good-quality cRSS and a specific chromatin modification, H3K4me3, satisfy the conditions of RAG's off-target effects on IKZF1. Our work provides evidence for RAG-mediated IKZF1 Δ3-6 deletion. Our results raise the prospect that RAG is a valuable biomarker in disease surveillance. Dissecting the contribution of RAG should not only provide valuable mechanistic insights, but will also lead to a new therapeutic direction.

Abstract A180: Topologically associated domains genome-wide restrict the off-target activity of recombination activating gene 1/2 endonuclease

Abstract Recombination activating gene 1/2 endonuclease (RAG) initiates V(D)J recombination in developing B and T lymphocytes via “on-target” activity at bona fide recombination signal sequences (RSSs) to generate diverse antibodies. “Off-target” RAG activity is thought to be rare, but can cause oncogenic interstitial deletions or chromosomal translocations that contribute to immature B and T lymphocyte cancers. Given that potential RAG off-target sites occur frequently in the genome, it has been a mystery how this potentially dangerous activity is suppressed. We generated mice with a pair of bona fide V(D)J RSSs (“V(D)J cassette”) in the first intron of c-myc on mouse chromosome 15. When introduced into an ATM-deficient background, the c-myc V(D)J cassette-containing mice recurrently develop B cell lymphomas with oncogenic c-myc translocations/amplifications involving the Igh locus on chromosome 12. These amplifications are generated by breakage-fusion-bridge (“BFB”) cycles from dicentric (15;12) chromosomes formed through fusion of RAG-generated Igh double-stranded breaks (DSBs) to sequences downstream of c-myc. Notably, the translocations downstream of c-myc occurred exclusively on V(D)J cassette-containing alleles, but did not involve the cassette. Based on this finding, we proposed that the V(D)J cassette mediates DSBs downstream of c-myc by activating RAG cutting at linked cryptic RSSs. We developed a linear amplification-mediated (“LAM”) version of our high-throughput genome-wide translocation sequencing approach (“LAM-HTGTS”) to robustly identify “prey” endogenous DNA DSBs that join to introduced or endogenous “bait” DSBs. We used LAM-HTGTS to reveal genome-wide DSBs that joined to RAG-generated bait DSBs. We found that translocations between RAG-induced DSBs in the c-myc V(D)J cassette to Igh occur only in the absence of ATM. Strikingly, however, we discovered a large number of deletions from the c-myc cassette to sequences just upstream of c-myc (1 kb) and up to 1.7 Mb downstream of c-myc in WT and ATM-deficient cells. Moreover, insertion of a pair of bona fide RSSs into 12 random sites genome-wide induced large numbers of RAG-initiated DSBs leading to cassette involved deletions and inversions spreading upstream and downstream of a given cassette from 200 kb to over 2 Mb, respectively. In each case, regions of RAG off-target activity fell into a well-defined topologically associated domain (TAD) into which the cassette integrated. Such domains generally were flanked by convergent CTCF binding sites (CBEs), such as those our lab implicated in Igh locus V(D)J recombination control, but sometimes also involved sub-domains of a CTCF-defined TAD. We showed these TAD-associated DSBs to enrich at “cryptic RSSs” determined by conserved “CAC” motifs. We also captured these DSBs via LAM-HTGTS from Cas9/gRNA DSBs within a TAD and proved them to be RAG-generated. We found dramatic changes of on-target profiles and expansion of off-target RAG-generated Igh locus DSBs when CBEs acting as boundary of DH-JH recombination domain were deleted. Based on these overall findings, we propose that antigen receptor loci exploit fixed CBE-based TADs to restrict RAG activity within them, but also evolved “regulated” sub-domains to further focus and re-focus RAG activity developmentally. We also used LAM-HTGTS to identify 89 RAG off-targets that participate in chromosomal translocations and show RAG off-targets occur in pairs in highly accessible regions such as enhancers and promoters within a TAD. Beyond providing a mechanisms for the generation of recurrent B cell lymphomas in ATM-deficient mice with a V(D)J cassette in c-myc, our findings help explain the origin of certain recurrent interstitial deletions and translocations in tumors that arise from human developing lymphocytes. Citation Format: Jiazhi Hu, Yu Zhang, Lijuan Zhao, Richard Frock, Zhou Du, Robin M. Meyers, Feilong Meng, David G. Schatz, Frederick W. Alt. Topologically associated domains genome-wide restrict the off-target activity of recombination activating gene 1/2 endonuclease. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A180.

Recruitment of RAG1 and RAG2 to Chromatinized DNA during V(D)J Recombination.

V(D)J recombination is initiated by the binding of the RAG1 and RAG2 proteins to recombination signal sequences (RSSs) that consist of conserved heptamer and nonamer sequences separated by a spacer of either 12 or 23 bp. Here, we used RAG-inducible pro-B v-Abl cell lines in conjunction with chromatin immunoprecipitation to better understand the protein and RSS requirements for RAG recruitment to chromatin. Using a catalytic mutant form of RAG1 to prevent recombination, we did not observe cooperation between RAG1 and RAG2 in their recruitment to endogenous Jκ gene segments over a 48-h time course. Using retroviral recombination substrates, we found that RAG1 was recruited inefficiently to substrates lacking an RSS or containing a single RSS, better to substrates with two 12-bp RSSs (12RSSs) or two 23-bp RSSs (23RSSs), and more efficiently to a substrate with a 12/23RSS pair. RSS mutagenesis demonstrated a major role for the nonamer element in RAG1 binding, and correspondingly, a cryptic RSS consisting of a repeat of CA dinucleotides, which poorly re-creates the nonamer, was ineffective in recruiting RAG1. Our findings suggest that 12RSS-23RSS cooperation (the "12/23 rule") is important not only for regulating RAG-mediated DNA cleavage but also for the efficiency of RAG recruitment to chromatin.

VH replacement in primary immunoglobulin repertoire diversification

The genes encoding the variable (V) region of the B-cell antigen receptor (BCR) are assembled from V, D (diversity), and J (joining) elements through a RAG-mediated recombination process that relies on the recognition of recombination signal sequences (RSSs) flanking the individual elements. Secondary V(D)J rearrangement modifies the original Ig rearrangement if a nonproductive original joint is formed, as a response to inappropriate signaling from a self-reactive BCR, or as part of a stochastic mechanism to further diversify the Ig repertoire. VH replacement represents a RAG-mediated secondary rearrangement in which an upstream VH element recombines with a rearranged VHDHJH joint to generate a new BCR specificity. The rearrangement occurs between the cryptic RSS of the original VH element and the conventional RSS of the invading VH gene, leaving behind a footprint of up to five base pairs (bps) of the original VH gene that is often further obscured by exonuclease activity and N-nucleotide addition. We have previously demonstrated that VH replacement can efficiently rescue the development of B cells that have acquired two nonproductive heavy chain (IgH) rearrangements. Here we describe a novel knock-in mouse model in which the prerearranged IgH locus resembles an endogenously rearranged productive VHDHJH allele. Using this mouse model, we characterized the role of VH replacement in the diversification of the primary Ig repertoire through the modification of productive VHDHJH rearrangements. Our results indicate that VH replacement occurs before Ig light chain rearrangement and thus is not involved in the editing of self-reactive antibodies.

PTEN microdeletions in T-cell acute lymphoblastic leukemia are caused by illegitimate RAG-mediated recombination events

AbstractPhosphatase and tensin homolog (PTEN)-inactivating mutations and/or deletions are an independent risk factor for relapse of T-cell acute lymphoblastic leukemia (T-ALL) patients treated on Dutch Childhood Oncology Group or German Cooperative Study Group for Childhood Acute Lymphoblastic Leukemia protocols. Some monoallelic mutated or PTEN wild-type patients lack PTEN protein, implying that additional PTEN inactivation mechanisms exist. We show that PTEN is inactivated by small deletions affecting a few exons in 8% of pediatric T-ALL patients. These microdeletions were clonal in 3% and subclonal in 5% of patients. Conserved deletion breakpoints are flanked by cryptic recombination signal sequences (cRSSs) and frequently have non-template-derived nucleotides inserted in between breakpoints, pointing to an illegitimate RAG recombination-driven activity. Identified cRSSs drive RAG-dependent recombination in a reporter system as efficiently as bona fide RSSs that flank gene segments of the T-cell receptor locus. Remarkably, equivalent microdeletions were detected in thymocytes of healthy individuals. Microdeletions strongly associate with the TALLMO subtype characterized by TAL1 or LMO2 rearrangements. Primary and secondary xenotransplantation of TAL1-rearranged leukemia allowed development of leukemic subclones with newly acquired PTEN microdeletions. Ongoing RAG activity may therefore actively contribute to the acquisition of preleukemic hits, clonal diversification, and disease progression.

VH Replacement Footprint Analyzer-I, a Java-Based Computer Program for Analyses of Immunoglobulin Heavy Chain Genes and Potential VH Replacement Products in Human and Mouse

VH replacement occurs through RAG-mediated secondary recombination between a rearranged VH gene and an upstream unrearranged VH gene. Due to the location of the cryptic recombination signal sequence (cRSS, TACTGTG) at the 3′ end of VH gene coding region, a short stretch of nucleotides from the previous rearranged VH gene can be retained in the newly formed VH–DH junction as a “footprint” of VH replacement. Such footprints can be used as markers to identify Ig heavy chain (IgH) genes potentially generated through VH replacement. To explore the contribution of VH replacement products to the antibody repertoire, we developed a Java-based computer program, VH replacement footprint analyzer-I (VHRFA-I), to analyze published or newly obtained IgH genes from human or mouse. The VHRFA-1 program has multiple functional modules: it first uses service provided by the IMGT/V-QUEST program to assign potential VH, DH, and JH germline genes; then, it searches for VH replacement footprint motifs within the VH–DH junction (N1) regions of IgH gene sequences to identify potential VH replacement products; it can also analyze the frequencies of VH replacement products in correlation with publications, keywords, or VH, DH, and JH gene usages, and mutation status; it can further analyze the amino acid usages encoded by the identified VH replacement footprints. In summary, this program provides a useful computation tool for exploring the biological significance of VH replacement products in human and mouse.

Genetic and epigenetic determinants mediate proneness of oncogene breakpoint sites for involvement in TCR translocations

T-cell receptor (TCR) translocations are a genetic hallmark of T-cell acute lymphoblastic leukemia and lead to juxtaposition of oncogene and TCR loci. Oncogene loci become involved in translocations because they are accessible to the V(D)J recombination machinery. Such accessibility is predicted at cryptic recombination signal sequence (cRSS) sites ('Type 1') as well as other sites that are subject to DNA double-strand breaks (DSBs) ('Type 2') during early stages of thymocyte development. As chromatin accessibility markers have not been analyzed in the context of TCR-associated translocations, various genetic and epigenetic determinants of LMO2, TAL1 and TLX1 translocation breakpoint (BP) sites and BP cluster regions (BCRs) were examined in human thymocytes to establish DSB proneness and heterogeneity of BP site involvement in TCR translocations. Our data show that DSBs in BCRs are primarily induced in the presence of a genetic element of sequence vulnerability (cRSSs, transposable elements), whereas breaks at single BP sites lacking such elements are more likely induced by chance or perhaps because of patient-specific genetic vulnerability. Vulnerability to obtain DSBs is increased by features that determine chromatin organization, such as methylation status and nucleosome occupancy, although at different levels at different BP sites.

NuChart: An R Package to Study Gene Spatial Neighbourhoods with Multi-Omics Annotations

Long-range chromosomal associations between genomic regions, and their repositioning in the 3D space of the nucleus, are now considered to be key contributors to the regulation of gene expression and important links have been highlighted with other genomic features involved in DNA rearrangements. Recent Chromosome Conformation Capture (3C) measurements performed with high throughput sequencing (Hi-C) and molecular dynamics studies show that there is a large correlation between colocalization and coregulation of genes, but these important researches are hampered by the lack of biologists-friendly analysis and visualisation software. Here, we describe NuChart, an R package that allows the user to annotate and statistically analyse a list of input genes with information relying on Hi-C data, integrating knowledge about genomic features that are involved in the chromosome spatial organization. NuChart works directly with sequenced reads to identify the related Hi-C fragments, with the aim of creating gene-centric neighbourhood graphs on which multi-omics features can be mapped. Predictions about CTCF binding sites, isochores and cryptic Recombination Signal Sequences are provided directly with the package for mapping, although other annotation data in bed format can be used (such as methylation profiles and histone patterns). Gene expression data can be automatically retrieved and processed from the Gene Expression Omnibus and ArrayExpress repositories to highlight the expression profile of genes in the identified neighbourhood. Moreover, statistical inferences about the graph structure and correlations between its topology and multi-omics features can be performed using Exponential-family Random Graph Models. The Hi-C fragment visualisation provided by NuChart allows the comparisons of cells in different conditions, thus providing the possibility of novel biomarkers identification. NuChart is compliant with the Bioconductor standard and it is freely available at ftp://fileserver.itb.cnr.it/nuchart.

Contribution of VH Replacement Products in Mouse Antibody Repertoire

VH replacement occurs through RAG-mediated recombination between the cryptic recombination signal sequence (cRSS) near the 3′ end of a rearranged VH gene and the 23-bp RSS from an upstream unrearranged VH gene. Due to the location of the cRSS, VH replacement leaves a short stretch of nucleotides from the previously rearranged VH gene at the newly formed V-D junction, which can be used as a marker to identify VH replacement products. To determine the contribution of VH replacement products to mouse antibody repertoire, we developed a Java-based VH Replacement Footprint Analyzer (VHRFA) program and analyzed 17,179 mouse IgH gene sequences from the NCBI database to identify VH replacement products. The overall frequency of VH replacement products in these IgH genes is 5.29% based on the identification of pentameric VH replacement footprints at their V-D junctions. The identified VH replacement products are distributed similarly in IgH genes using most families of VH genes, although different families of VH genes are used differentially. The frequencies of VH replacement products are significantly elevated in IgH genes derived from several strains of autoimmune prone mice and in IgH genes encoding autoantibodies. Moreover, the identified VH replacement footprints in IgH genes from autoimmune prone mice or IgH genes encoding autoantibodies preferentially encode positively charged amino acids. These results revealed a significant contribution of VH replacement products to the diversification of antibody repertoire and potentially, to the generation of autoantibodies in mice.

V(D)J Recombinase-Mediated TCR β Locus Gene Usage and Coding Joint Processing in Peripheral T Cells during Perinatal and Pediatric Development

The generation of TCR proteins is the result of V(D)J recombinase-mediated genomic rearrangements at recombination signal sequences (RSS) in human lymphocytes. V(D)J recombinase can also mediate rearrangements at nonimmune or "cryptic" RSS in normal and leukemic human peripheral T cells. We previously demonstrated age- and gender-specific developmental differences in V(D)J coding joint processing at cryptic RSS within the HPRT locus in peripheral T cells from healthy children (Murray et al. 2006. J. Immunol. 177: 5393-5404). In this study, we investigated developmentally specific V(D)J recombinase TCRβ immune gene rearrangements and coding joint processing at RSS in peripheral T cells in the same pediatric population. This approach provided a unique opportunity to investigate site-specific V(D)J recombinase rearrangements and coding joint processing at immune and nonimmune genes from the same individual T cell population. We determined the genomic sequence of 244 TCRβ coding junctions from 112 (63 male, 49 female) subjects from the late stages of fetal development through 9 y of age. We observed both age- and gender-specific V(D)J recombinase-mediated TCRβ gene usage and coding joint processing at immune RSS. To the best of our knowledge, these data represent the first description of age- and gender-specific developmental differences in TCR gene usage and coding joint processing that could directly influence TCR diversity and immune specificity. It will be important for future studies to ascertain the mechanistic etiology of these developmental and gender differences in TCR diversity and specificity, as well as their importance with respect to the age and gender risks for infectious and autoimmune diseases in humans.

Partial organization of bovine variable-heavy chain gene locus and influence of recombination signal sequences (RSS) on various variable region gene expression (42.6)

Abstract An attempt has been made to determine the organization of variable-heavy chain gene locus in cattle, based on 91% assembled Hereford cow genome sequence, where both functional VH and pseudogenes are identified on chromosome 7 and 21. Of a total of 13 VH genes identified unambiguously, 6 VH genes are located on chromosome 21 and 7 VH genes are present on chromosome 7. Interestingly, the functional VH genes are distributed on both chromosome 7 and 21 raising questions about VDJ recombination occurring on two different chromosomes (Hosseini, A. et al. 2004. Int. Immunol.16:843) or via as yet unidentified mechanism that possibly permitted allelic exclusion. The presence of 10 DH-genes in the Hereford genome is confirmed as earlier reported by our laboratory (Koti, M. et al. 2010. Mol Immunol. 47:2119). Come cryptic recombination signal sequences (RSS) seem to exist within the VH encoding region requiring elucidation of their role in VDJ recombination. Nevertheless, V-gene expression in bovine B cells is influenced by the composition of RSSs. These findings, especially the presence of variable heavy chain locus on two chromosomes, raise fundamental questions about the phenomenon and mechanisms of allelic exclusion in bovine B cells.

Spatiotemporal analysis of illegitimate V(D)J deletions in Notch1 and Bcl11b genes (162.14)

Abstract Aberrant V(D)J recombination events in oncogenes and tumor suppressor genes are implicated in development of T-cell leukemias and lymphomas. Previously, others identified hotspots for deletional DNA rearrangements adjacent to cryptic recombination signal sequences in Notch1 and Bcl11b genes in thymus of adult mice with radiation-induced thymic lymphomas. Interestingly, these rearrangements were also found at detectable frequencies in thymus of unirradiated WT animals suggesting that factors in addition to deletions contribute to development of lymphomas, but the frequency of deletions at different developmental stages or in peripheral lymphoid organs was not analyzed. To determine the timing, frequency, and tissue distribution of Notch1 and Bcl11b deletions during development, we are examining tissues harvested at different developmental stages from C57BL/6 mice. We detect these deletions in thymus at all ages examined from fetus to 6 months. Deletions are also detected in spleen, consistent with the presence of T cells bearing deletions in the circulating lymphocyte pool. We are analyzing sequences of deletion junctions for clonal relationships in individual animals and performing broken DNA end analysis for ongoing deletions. We are also using an inducible tetracycline-regulated RAG transgenic mouse system to determine if these rearrangements can occur at all ages with induced RAG expression or are limited to specific developmental windows.

Phylogenetic conservation of the 3′ cryptic recombination signal sequence (3′cRSS) in the VH genes of jawed vertebrates

The VH replacement process is a RAG-mediated secondary recombination in which the variable region of a rearranged VHDJH is replaced by a different germline VH gene. In almost all human and mouse VH genes, two sequence features appear to be crucial for VH replacement. First, an embedded heptamer, which is located near the 3′ end of the rearranged VH gene, serves as a cryptic recombination signal sequence (3′cRSS) for the VH replacement process. Second, a short stretch of nucleotides located downstream of the 3′cRSS serve as a footprint of the original VH region, frequently encoding charged amino acids. In this review, we show that both of these two features are conserved in the VH genes of all jawed vertebrates, which suggests that the VH replacement process may be a conserved mechanism.