Sequence-dependent dynamics of synthetic and endogenous RSSs in V(D)J recombination

Hirokawa, Soichi; Chure, Griffin; Belliveau, Nathan M.; Lovely, Geoffrey A.; Anaya, Michael; Schatz, David G.; Baltimore, David; Phillips, Rob

doi:10.1093/nar/gkaa418

Cited by 12 publications

(12 citation statements)

References 59 publications

(95 reference statements)

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“…2 B and C). Consistent with a previous biochemical study, G is least preferred at position 6 (23), and >80% of RSS heptamers containing G in this position are in the bottom fourth of RSSs reproducibly utilized in the SARP-seq method (Fig. 2B and Supplementary Dataset S1).…”

Section: The R/y Pattern In the Rss Heptamer Is Highly Predictive Of Rag1/2 Activitysupporting

confidence: 90%

High Throughput Characterization of V(D)J Recombination Signal Sequences Redefines the Consensus Sequence

Hoolehan

Harris

Byrum

et al. 2021

Preprint

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In the adaptive immune system, V(D)J recombination initiates the production of a diverse antigen receptor repertoire in developing B and T cells. Recombination activating proteins, RAG1 and RAG2 (RAG1/2), catalyze V(D)J recombination by cleaving adjacent to recombination signal sequences (RSSs) that flank antigen receptor gene segments. Previous studies defined the consensus RSS as containing conserved heptamer and nonamer sequences separated by a less conserved 12 or 23 base-pair spacer sequence. However, many RSSs deviate from the consensus sequence. Here, we developed a cell-based, massively parallel V(D)J recombination assay to evaluate RAG1/2 activity on thousands of RSSs. We focused our study on the RSS heptamer and adjoining spacer region, as this region undergoes extensive conformational changes during RAG-mediated DNA cleavage. While the consensus heptamer sequence (CACAGTG) was marginally preferred, RAG1/2 was highly active on a wide range of non-consensus sequences. RAG1/2 generally preferred select purine/pyrimidine motifs that may accommodate heptamer unwinding in the RAG1/2 active site. Our results suggest RAG1/2 specificity for RSS heptamers is primarily dictated by DNA structural features dependent on purine/pyrimidine pattern, and to a lesser extent, RAG:RSS base-specific interactions. Further investigation of RAG1/2 specificity using this new approach will help elucidate the genetic instructions guiding V(D)J recombination.Summary StatementPartially conserved recombination signal sequences (RSSs) govern antigen receptor gene assembly during V(D)J recombination. Here, a massively parallel analysis of randomized RSSs reveals key attributes that allow DNA sequence diversity in the RAG1/2 active site and that contribute to the differential utilization of RSSs in endogenous V(D)J recombination. Overall, these results will assist identification of RAG1/2 off-target sites, which can drive leukemia cell transformation, as well as characterization of bona fide RSSs used to generate antigen receptor diversity.

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Section: The R/y Pattern In the Rss Heptamer Is Highly Predictive Of Rag1/2 Activitysupporting

confidence: 90%

High Throughput Characterization of V(D)J Recombination Signal Sequences Redefines the Consensus Sequence

Hoolehan

Harris

Byrum

et al. 2021

Preprint

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“…Another shortcoming of the current implementation of the method is that it misses regulatory action at a distance. Indeed, our laboratory has invested a significant effort in exploring such long-distance regulatory action in the form of DNA looping in bacteria ( Johnson et al, 2012 ; Han et al, 2009 ) and V(D)J recombination in jawed vertebrates ( Lovely et al, 2015 ; Hirokawa et al, 2020 ). It is well known that transcriptional control through enhancers in eukaryotic regulation is central in contexts ranging from embryonic development to hematopoiesis ( Melnikov et al, 2012 ).…”

Section: Discussionmentioning

confidence: 99%

Deciphering the regulatory genome of Escherichia coli, one hundred promoters at a time

Ireland

Beeler

Flores-Bautista

et al. 2020

eLife

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Advances in DNA sequencing have revolutionized our ability to read genomes. However, even in the most well-studied of organisms, the bacterium Escherichia coli, for ≈ 65% of promoters we remain ignorant of their regulation. Until we crack this regulatory Rosetta Stone, efforts to read and write genomes will remain haphazard. We introduce a new method, Reg-Seq, that links massively-parallel reporter assays with mass spectrometry to produce a base pair resolution dissection of more than 100 E. coli promoters in 12 growth conditions. We demonstrate that the method recapitulates known regulatory information. Then, we examine regulatory architectures for more than 80 promoters which previously had no known regulatory information. In many cases, we also identify which transcription factors mediate their regulation. This method clears a path for highly multiplexed investigations of the regulatory genome of model organisms, with the potential of moving to an array of microbes of ecological and medical relevance.

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“…Recently, a research reported cryoelectron microscopy structures of synaptic RAG complexes at up to 3.4 Å resolution, which reveal a closed conformation with base flipping and base-specific recognition of RSSs ( Ru et al, 2015 ). Another study employed a single-molecule method to track the RAG–RSS interaction, which can provide a relatived complete kinetic description of the initial phases of V(D)J recombination ( Hirokawa et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

The Usage of Human IGHJ Genes Follows a Particular Non-random Selection: The Recombination Signal Sequence May Affect the Usage of Human IGHJ Genes

Shu

Dong

et al. 2020

Front. Genet.

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The formation of the B cell receptor (BCR) heavy chain variable region is derived from the germline V(D)J gene rearrangement according to the “12/23” rule and the “beyond 12/23” rule. The usage frequency of each V(D)J gene in the peripheral BCR repertoires is related to the initial recombination, self-tolerance selection, and the clonal proliferative response. However, their specific differences and possible mechanisms are still unknown. We analyzed in-frame and out-of-frame BCR-H repertoires from human samples with normal physiological and various pathological conditions by high-throughput sequencing. Our results showed that IGHJ gene frequency follows a similar pattern which is previously known, where IGHJ4 is used at high frequency (>40%), IGHJ6/IGHJ3/IGHJ5 is used at medium frequencies (10∼20%), and IGH2/IGHJ1 is used at low frequency (<4%) under whether normal physiological or various pathological conditions. However, our analysis of the recombination signal sequences suggested that the conserved non-amer and heptamer and certain 23 bp spacer length may affect the initial IGHD-IGHJ recombination, which results in different frequencies of IGHJ genes among the initial BCR-H repertoire. Based on this “initial repertoire,” we recommend that re-evaluation and further investigation are needed when analyzing the significance and mechanism of IGHJ gene frequency in self-tolerance selection and the clonal proliferative response.

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Sequence-dependent dynamics of synthetic and endogenous RSSs in V(D)J recombination

Cited by 12 publications

References 59 publications

High Throughput Characterization of V(D)J Recombination Signal Sequences Redefines the Consensus Sequence

High Throughput Characterization of V(D)J Recombination Signal Sequences Redefines the Consensus Sequence

Deciphering the regulatory genome of Escherichia coli, one hundred promoters at a time

The Usage of Human IGHJ Genes Follows a Particular Non-random Selection: The Recombination Signal Sequence May Affect the Usage of Human IGHJ Genes

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