VDJ RECOMBINATION ACTIVATING GENES--FUNCTIONAL ANALYSIS

Summary

Principal Investigator: Marjorie Oettinger
Affiliation: Harvard University
Country: USA
Abstract: A central process in the development of a competent immune system is the series of genomic rearrangement events that take place to produce a mature, functional immunoglobulin or T cell receptor gene. This complex process, known as V(D)J recombination, has been studied intensively since its discovery in 1975, but the reaction and enzymatic machinery involved in it remain poorly understood. We have recently identified two genes, RAG-1 and RAG-2, that are capable of inducing this lymphoid specific event in non- lymphoid cells (i.e. fibroblasts) and are extremely likely to encode part of the V(D)J recombinase machinery. These genes provide new tools with which to probe the events surrounding the rearrangement process. Our goal over the next years is to understand the function(s) of the RAG genes and through them to gain a greater understanding of how the rearrangement events are carried out and regulated. First, we will conduct a mutational analysis of RAG-1 and RAG-2 to determine the minimum functional sequence required for recombinase activity and to functionally dissect the RAG proteins. Deletion and point mutations will be analyzed using a number of standard assays for recombinase activity to determine if any of the mutants are defective in particular aspects of the reaction. Second, we will attempt to develop new assays with which to identify partial reaction products of the V(D)J recombination reaction. In developing these assays we will make use of the mutants described above and of our ability to control where and when the RAG genes and recombinase activity are expressed. Third, both RAG-1 and RAG-2 are required for the induction of V(D)J recombination. Thus, we have designed genetic and biochemical experiments to test whether these proteins interact with each other, or if either protein multimerizes. If interactions are detected, we will determine the portions of the protein(s) required for the interactions. Further, we will attempt to define other factors with which RAG-1 and/or RAG-2 interact. The identification of such factors would be important for our understanding of the events involved in V(D)J recombination. Fourth, we will use both genetic and biochemical approaches in order to test RAG-1 and RAG-2 for a number of enzymatic activities (alone and in combination) that are expected properties of the V(D)J recombinase. Furthermore, because we have demonstrated a correlation between RAG-2 expression and gene conversion activity, we will test the hypothesis that there is a causal relationship between the two. In summary, developing lymphocytes must assemble their antigen receptor genes; failure to do so can result in immunodeficiency. The proposed experiments should provide information on the structure and function(s) of RAG-1 and RAG-2, two genes that appear to play a critical role in V(D)J recombination. Further, this work should lead to a better understanding of how this exceedingly important and complex reaction is carried out.
Funding Period: 1992-08-01 - 1997-07-31
more information: NIH RePORT

Top Publications

  1. pmc RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination
    Adam G W Matthews
    Department of Molecular Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA
    Nature 450:1106-10. 2007
  2. pmc Mice lacking Sμ tandem repeats maintain RNA polymerase patterns but exhibit histone modification pattern shifts linked to class switch site locations
    Barbara B Balter
    Immunology Program and Department of Pathology, Tufts University School of Medicine, Boston, MA 02111, USA
    Mol Immunol 52:1-8. 2012
  3. pmc Histone H3R2 symmetric dimethylation and histone H3K4 trimethylation are tightly correlated in eukaryotic genomes
    Chih Chi Yuan
    Department of Molecular Biology, Massachusetts General Hospital and Department of Genetics, Harvard Medical School, Boston, MA 02114, USA
    Cell Rep 1:83-90. 2012
  4. pmc RAG: a recombinase diversified
    Adam G W Matthews
    Howard Hughes Medical Institute, Department of Biology, Cambridge, Massachusetts, USA
    Nat Immunol 10:817-21. 2009
  5. ncbi Regulation of RAG transposition
    Adam G W Matthews
    Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA
    Adv Exp Med Biol 650:16-31. 2009
  6. ncbi The MRE11-RAD50-XRS2 complex, in addition to other non-homologous end-joining factors, is required for V(D)J joining in yeast
    Anne E Clatworthy
    Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA
    J Biol Chem 280:20247-52. 2005
  7. pmc Determinants of HMGB proteins required to promote RAG1/2-recombination signal sequence complex assembly and catalysis during V(D)J recombination
    Yan Dai
    Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA
    Mol Cell Biol 25:4413-25. 2005
  8. ncbi A PHD finger motif in the C terminus of RAG2 modulates recombination activity
    Sheryl K Elkin
    Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA
    J Biol Chem 280:28701-10. 2005

Scientific Experts

  • Adam G W Matthews
  • Marjorie A Oettinger
  • Chih Chi Yuan
  • Barbara B Balter
  • Anne E Clatworthy
  • Sheryl K Elkin
  • Yan Dai
  • Yi Jin
  • Tatiana G Kutateladze
  • Mark L Borowsky
  • Brad A Chapman
  • Karen C Glass
  • Erik Selsing
  • Toshiro K Ohsumi
  • David N Ciccone
  • Chang Feng Chen
  • Kevin Struhl
  • Mark Ewalt
  • Glenn D Prestwich
  • Sven G Hyberts
  • Colin G Ferguson
  • Dmitri Ivanov
  • Zhen Yu J Sun
  • Junying Yuan
  • Yi Meng Yen
  • Gerhard Wagner
  • Maria A Valencia-Burton
  • Ben Wong
  • Jongbum Kwon
  • James E Haber
  • Or P Gozani
  • Reid C Johnson

Detail Information

Publications8

  1. pmc RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination
    Adam G W Matthews
    Department of Molecular Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA
    Nature 450:1106-10. 2007
    ..Furthermore, our results provide the first evidence indicating that disrupting the read-out of histone modifications can cause an inherited human disease...
  2. pmc Mice lacking Sμ tandem repeats maintain RNA polymerase patterns but exhibit histone modification pattern shifts linked to class switch site locations
    Barbara B Balter
    Immunology Program and Department of Pathology, Tufts University School of Medicine, Boston, MA 02111, USA
    Mol Immunol 52:1-8. 2012
    ....
  3. pmc Histone H3R2 symmetric dimethylation and histone H3K4 trimethylation are tightly correlated in eukaryotic genomes
    Chih Chi Yuan
    Department of Molecular Biology, Massachusetts General Hospital and Department of Genetics, Harvard Medical School, Boston, MA 02114, USA
    Cell Rep 1:83-90. 2012
    ..Our work suggests that H3R2me2sK4me3, not simply H3K4me3 alone, is the mark of active promoters and that factors that recognize H3K4me3 will have their binding modulated by their preference for H3R2me2s...
  4. pmc RAG: a recombinase diversified
    Adam G W Matthews
    Howard Hughes Medical Institute, Department of Biology, Cambridge, Massachusetts, USA
    Nat Immunol 10:817-21. 2009
    ..Here we discuss some of these functions and suggest that the RAG-1-RAG-2 complex nucleates a specialized subnuclear compartment that we call the 'V(D)J recombination factory'...
  5. ncbi Regulation of RAG transposition
    Adam G W Matthews
    Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, USA
    Adv Exp Med Biol 650:16-31. 2009
    ..We then discuss the discovery of RAG transposition and present an overview of the RAG transposition pathway. Using this pathway as a framework, we discuss the factors and forces that regulate RAG transposition...
  6. ncbi The MRE11-RAD50-XRS2 complex, in addition to other non-homologous end-joining factors, is required for V(D)J joining in yeast
    Anne E Clatworthy
    Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA
    J Biol Chem 280:20247-52. 2005
    ..In addition, we showed an absolute requirement for the MRX complex in signal joining, suggesting that the Mre11-Rad50-Nbs1 complex may be required for signal joint formation in mammalian cells as well...
  7. pmc Determinants of HMGB proteins required to promote RAG1/2-recombination signal sequence complex assembly and catalysis during V(D)J recombination
    Yan Dai
    Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA
    Mol Cell Biol 25:4413-25. 2005
    ..The resulting complex must be sufficiently dynamic to enable the series of RAG1/2-mediated chemical reactions on the DNA...
  8. ncbi A PHD finger motif in the C terminus of RAG2 modulates recombination activity
    Sheryl K Elkin
    Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA
    J Biol Chem 280:28701-10. 2005
    ..We propose a model in which the equilibrium between these states modulates recombination activity. Together, these data identify the PHD finger as a novel and functionally important domain of RAG2...