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Transcription factor Pax5 (BSAP) transactivates the RAG-mediated VH-to-DJH rearrangement of immunoglobulin genes

Nature Immunology volume 7, pages 616–624 (2006)Cite this article

Abstract

Immunoglobulin rearrangement from variable heavy chain (VH) to diversity (D)–joining heavy chain (JH), which occurs exclusively in B lineage cells, is impaired in mice deficient for the B lineage–specific transcription factor Pax5. Conversely, ectopic Pax5 expression in thymocytes promotes the rearrangement of DH-proximal VH7183 genes. In exploring the mechanism for Pax5 regulation of VH-to-DJH recombination, we have identified multiple Pax5 binding sites in the coding regions of human and mouse VH gene segments. Pax5 bound to those sites in vitro and occupied VH genes in early human and mouse B lineage cells. Moreover, Pax5 interacted with the recombination-activating gene 1 (RAG1)–RAG2 complex to enhance RAG-mediated VH recombination signal sequence cleavage and recombination of a VH gene substrate. These findings indicate a direct activating function for Pax5 in RAG-mediated immunoglobulin VH-to-DJH recombination.

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Figure 1: In vitro analysis of the association of Pax5 with VH coding regions.
Figure 2: Pax5 associates with VH coding regions in developing B lineage cells.
Figure 3: Interaction of Pax5 with RAG1 and RAG2 proteins.
Figure 4: Physical interaction of Pax5 with the RAG1-RAG2 complex.
Figure 5: Pax5 enhances RAG-mediated VH RSS cleavage.
Figure 6: Pax5 enhances RAG-mediated recombination of VH gene substrates.
Figure 7: Ectopic expression of Pax5, E47, RAG1 and RAG2 induces IGH@ VH-to-DJH recombination in 293T cells.

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Acknowledgements

We thank M. Busslinger (Research Institute of Molecular Pathology, Vienna, Austria) for the Pax5 expression vectors and discussions; D. Roth (Baylor College of Medicine, Houston, Texas), M. Gellert (National Institutes of Health, Bethesda, Maryland) and D. Schatz (Yale University, New Haven, Connecticut) for the pEBG-RAG1, pEBG-RAG2, pJH289 and pJH290 vectors, protocols for RAG protein purification and in vitro cleavage assays, and discussions; S. Desiderio (Johns Hopkins University, Baltimore, Maryland) for antibodies to RAG1 and RAG2, pCDNA1-RAG1 and pCDNA1-RAG2 expression vectors, and suggestions; Y. Kubagawa for DNA sequencing; and members of the laboratories of M.D.C., Z.Z., A.J.F. and J.H. for discussions. Supported by the National Institutes of Health (AI39816 and AI48098; AI56322, AI54661 and CA107478 to J.H.; and AI52313 to the laboratory of A.J.F.), the National Institutes of Health–National Institute of Arthritis, Musculoskeletal and Skin Diseases (K01 AR048592 to Z.Z.) and the Howard Hughes Medical Institute (M.D.C.).

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Authors and Affiliations

  1. Division of Developmental and Clinical Immunology, Birmingham, 35294, Alabama, USA

    Zhixin Zhang, Zhihong Yu, Robert Stephan, Ti He, Peter D Burrows & Max D Cooper

  2. Department of Medicine, Birmingham, 35294, Alabama, USA

    Zhixin Zhang, Zhihong Yu, Robert Stephan & Max D Cooper

  3. Department of Cell Biology, Birmingham, 35294, Alabama, USA

    Zhixin Zhang

  4. Department of Pediatrics, Birmingham, 35294, Alabama, USA

    Max D Cooper

  5. Department of Microbiology, Birmingham, 35294, Alabama, USA

    Zhixin Zhang, Ti He, Peter D Burrows & Max D Cooper

  6. Department of Genetics, Birmingham, 35294, Alabama, USA

    Peter D Burrows

  7. Department of Pathology, University of Alabama at Birmingham, Birmingham, 35294, Alabama, USA

    Max D Cooper

  8. Howard Hughes Medical Institute, Birmingham, 35294, Alabama, USA

    Max D Cooper

  9. Integrated Department of Immunology, National Jewish Medical and Research Center, Denver, 80206, Colorado, USA

    James Hagman

  10. Department of Immunology, The Scripps Research Institute, La Jolla, 92037, California, USA

    Celia R Espinoza, G Stuart Williams & Ann J Feeney

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Supplementary information

Supplementary Fig. 1

Sequence alignment of candidate Pax5 binding sites from the VH genes with the consensus Pax5 binding sites. (PDF 16 kb)

Supplementary Fig. 2

Pax5 association with the VH1-8-Pax5 binding sites. (PDF 114 kb)

Supplementary Fig. 3

Pax5 association with the individual mouse VH gene Pax5 binding sites. (PDF 338 kb)

Supplementary Fig. 4

Interaction of Pax5 with the RAG1/RAG2 complex. (PDF 103 kb)

Supplementary Fig. 5

Effects of Pax5 on RAG-mediated cleavage and recombination. (PDF 46 kb)

Supplementary Fig. 6

Effects of Gal4-Pax5-ΔB on RAG-mediated recombination of pJH298-G4RSS constructs. (PDF 36 kb)

Supplementary Table 1

Identification of potential Pax5 binding sites within human VH1 family of VH genes. (PDF 40 kb)

Supplementary Table 2

Identification of potential Pax5 binding sites within human VH3 family of VH genes. (PDF 48 kb)

Supplementary Table 3

Identification of potential Pax5 binding sites within human VH4 family of VH genes. (PDF 38 kb)

Supplementary Table 4

Identification of potential Pax5 binding sites within mouse VHJ558 family of VH genes. (PDF 119 kb)

Supplementary Table 5

Identification of potential Pax5 binding sites within mouse VH7183 family of VH genes. (PDF 62 kb)

Supplementary Table 6

Identification of potential Pax5 binding sites within mouse VHS107 family of VH genes. (PDF 40 kb)

Supplementary Table 7

Summary of potential Pax5 binding sites within human and mouse VH genes. (PDF 35 kb)

Supplementary Table 8

Sequence analysis of V(D)J joint obtained from 293T cells expressing RAG1, RAG2, E47, and Pax5. (PDF 43 kb)

Supplementary Table 9

List of oligonucleotide sequences. (PDF 78 kb)

Supplementary Methods (PDF 67 kb)

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Zhang, Z., Espinoza, C., Yu, Z. et al. Transcription factor Pax5 (BSAP) transactivates the RAG-mediated VH-to-DJH rearrangement of immunoglobulin genes. Nat Immunol 7, 616–624 (2006). https://doi.org/10.1038/ni1339

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