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The tissue- and stage-specific assembly of antigen receptor genes by V(D)J recombination is regulated by changes in the chromatin accessibility of target gene segments. This dynamic remodeling process is coordinated by cis-acting promoters and enhancers, which function as accessibility control elements. The basic epigenetic mechanisms that activate or repress chromatin accessibility to V(D)J recombinase remain unclear. We now demonstrate that a histone methyltransferase overrides accessibility control element function and cripples V(D)J recombination of chromosomal gene segments. The recruited histone methyltransferase induces extensive revisions in the local chromatin environment, including altered histone modifications and de novo methylation of DNA. These findings indicate a key function for histone methyltransferases in the tissue- and stage-specific suppression of antigen receptor gene assembly during lymphocyte development.

The assembly of genes encoding antigen receptors is regulated by developmental changes in chromatin that either permit or deny access to a single variable-(diversity)-joining recombinase. These changes are guided by transcriptional promoters and enhancers, which serve as accessibility-control elements in antigen-receptor loci. The function of each accessibility-control element and the factors they recruit to remodel chromatin remain obscure. Here we show that the recruitment of SWI-SNF chromatin-remodeling complexes compensated for the accessibility-control element function of a promoter but not an enhancer of the T cell receptor-beta locus (Tcrb). Loss of SWI-SNF function in thymocytes inactivated recombinase targets at the endogenous Tcrb locus. Thus, initiation of Tcrb gene assembly and T cell development is contingent on the recruitment of SWI-SNF to promoters, which exposes gene segments to variable-(diversity)-joining recombinase.

i V(D)J recombination is initiated by the RAG endonuclease, which introduces DNA double-strand breaks (DSBs) at the border between two recombining gene segments, generating two hairpin-sealed coding ends and two blunt signal ends. ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are serine-threonine kinases that orchestrate the cellular responses to DNA DSBs. During V(D)J recombination, ATM and DNA-PKcs have unique functions in the repair of coding DNA ends. ATM deficiency leads to instability of postcleavage complexes and the loss of coding ends from these complexes. DNA-PKcs deficiency leads to a nearly complete block in coding join formation, as DNA-PKcs is required to activate Artemis, the endonuclease that opens hairpin-sealed coding ends. In contrast to loss of DNA-PKcs protein, here we show that inhibition of DNA-PKcs kinase activity has no effect on coding join formation when ATM is present and its kinase activity is intact. The ability of ATM to compensate for DNA-PKcs kinase activity depends on the integrity of three threonines in DNA-PKcs that are phosphorylation targets of ATM, suggesting that ATM can modulate DNA-PKcs activity through direct phosphorylation of DNA-PKcs. Mutation of these threonine residues to alanine (DNA-PKcs 3A ) renders DNA-PKcs dependent on its intrinsic kinase activity during coding end joining, at a step downstream of opening hairpinsealed coding ends. Thus, DNA-PKcs has critical functions in coding end joining beyond promoting Artemis endonuclease activity, and these functions can be regulated redundantly by the kinase activity of either ATM or DNA-PKcs. Antigen receptor genes are assembled in developing lymphocytes through the process of V(D)J recombination (1). The V(D)J recombination reaction forms the second exon of these genes from component variable (V), joining (J), and, at some loci, diversity (D) gene segments. V(D)J recombination is initiated when the RAG-1 and RAG-2 proteins, which together form the RAG endonuclease, introduce DNA double-strand breaks (DSBs) at the border of two recombining gene segments and their associated RAG recognition sequences, termed recombination signals (RSs) (2). DNA cleavage by RAG results in two broken DNA ends with distinct structures: a blunt signal end and a coding end that is hairpin sealed by a phosphodiester bond connecting the top and bottom strands (2).

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