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Altered 3D chromatin structure permits inversional recombination at the IgH locus

Citation

Qiu, Xiang (2021), Altered 3D chromatin structure permits inversional recombination at the IgH locus, Dryad, Dataset, https://doi.org/10.5061/dryad.kprr4xh1s

Abstract

Immunoglobulin heavy chain (IgH) genes are assembled by two sequential DNA rearrangement events that are initiated by recombinase activating gene products (RAG) 1 and 2.  Diversity gene segments (DH) rearrange first, followed by variable (VH) gene rearrangements.  Here we provide evidence that each rearrangement step is guided by different rules of engagement between rearranging gene segments.  DH gene segments, that recombine by deletion of intervening DNA, must be located within a RAG1/2 scanning domain for efficient recombination.  In the absence of intergenic control region 1, a regulatory sequence that delineates the RAG scanning domain on WT IgH alleles, VH and DH gene segments can recombine with each other by both deletion and inversion of intervening DNA.  We propose that VH gene segments find their targets by diffusion-controlled mechanisms.  These distinct mechanisms may underlie differential allelic choice associated with each step of IgH gene assembly.

Methods

DJH/VDH recombination assays were carried out as previously described.  Genomic DNA was purified from sorted bone marrow pro-B cells (IgM-B220+CD43+) from WT or IGCR1-/- mice.  5-fold serial dilutions of genomic DNA (200ng, 40ng, 8ng) were used to perform PCR to analyze DJH rearrangements.  Primers used in this assay are listed in Table S5.  Primers flanking the ROSA26 gene were used as a loading control under the same conditions.  GeneRuler 100 bp Plus (Thermo Fisher Scientific, #SM0324) was used to confirm sizes of PCR products.

RAG2-deficient pro-B cell lines, CBE-/-(1), CBE-/-(2), WT, CBE-/-(1)/C1-/-#1, CBE-/-(1)/C1-/- #2, and CBE-/-(1)/C1-/-C2-/-, were infected with RAG2-expressing lentivirus, and cultured in complete medium with puromycin (Thermo Fisher Scientific, #A1113803, 2μg/ml).  After 14 days selection with puromycin, cells were harvested.  Genomic DNA was collected with DNeasy Blood & Tissue Kit (Qiagen, #69506), and the DNA was used to analyze DJH/VDJH rearrangements with HotStarTaq DNA Polymerase (Qiagen, #203205) as described as above. Amplification products were analyzed by agarose gel electrophoresis. 

Usage Notes

Raw images are corresponded to Figures 2, 3, 5, S2 and S5 in the manuscript.

Funding

National Institutes of Health, Award: AI079002 (to MA)

National Institutes of Health, Award: GM111384 (to MA)

National Institutes of Health, Award: Intramural Research Program (to RS)