Article

The In Vivo Pattern of Binding of RAG1 and RAG2 to Antigen Receptor Loci (vol 141, pg 419, 2010)

Department of Immunobiology, Yale University School of Medicine, 300 Cedar Street, Box 208011, New Haven, CT 06520-8011, USA.
Cell (Impact Factor: 32.24). 04/2010; 141(3):419-31. DOI: 10.1016/j.cell.2010.03.010
Source: PubMed

ABSTRACT

The critical initial step in V(D)J recombination, binding of RAG1 and RAG2 to recombination signal sequences flanking antigen receptor V, D, and J gene segments, has not previously been characterized in vivo. Here, we demonstrate that RAG protein binding occurs in a highly focal manner to a small region of active chromatin encompassing Ig kappa and Tcr alpha J gene segments and Igh and Tcr beta J and J-proximal D gene segments. Formation of these small RAG-bound regions, which we refer to as recombination centers, occurs in a developmental stage- and lineage-specific manner. Each RAG protein is independently capable of specific binding within recombination centers. While RAG1 binding was detected only at regions containing recombination signal sequences, RAG2 binds at thousands of sites in the genome containing histone 3 trimethylated at lysine 4. We propose that recombination centers coordinate V(D)J recombination by providing discrete sites within which gene segments are captured for recombination.

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    • "In summary, we propose that the-deficient pro-B cells were kindly provided by Dr. J. Pongubala. The Abelson-MuLV-transformed pro-B cell line was D345 (Ji et al., 2010). FISH was performed as described (Guo et al., 2011a). "
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    • "Our findings at naturally occurring translocation sites extend recent correlative observations from genome-wide studies that mapped I-SceI-induced translocation junctions in B cells to within or near transcriptionally active regions of the genome (Chiarle et al. 2011; Klein et al. 2011) and in translocation-negative prostate cancer cells, where liganded androgen receptor (AR) binds near the TMPRSS2 translocation gene and increases its transcriptional activity, leading to elevated H3K4me3 levels across the breakpoint region (Lin et al. 2009; Yu et al. 2010). Furthermore, H3K4me3 has been implicated in DSBs generated by endonucleases during class switch and V(D)J recombination processes in lymphocytes (Daniel and Nussenzweig 2012), and both RAG2 and AID target extrachromosomal sites that are highly enriched in H3K4me3 in a sequence-independent manner (Shimazaki et al. 2009; Ji et al. 2010; Stanlie et al. 2010). The increased formation of DSBs by endonucleases in regions of high H3K4 methylation is likely due to chromatin decondensation, resulting in higher chromatin accessibility (Mostoslavsky et al. 2003). "
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    • "Green shading indicates whether cross-linking in mE relative to WT alleles is unchanged (darkest green), reduced significantly (lighter green), or reduced to background levels in pro-B cells (white). (F) 3C assays were performed with the E viewpoint (anchor) in DN thymocytes and pro-B cells from RAG-deficient mice, either lacking or expressing a D708A RAG transgene (Ji et al., 2010b). (G) 3C assays were performed with the D1 viewpoint (anchor) in DN thymocytes (WT, E, or E alleles) and pro-B cell controls. "
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