Flexible ordering of antibody class switch and V(D)J joining during B-cell ontogeny.

V(D)J joining is mediated by RAG recombinase during early B-lymphocyte development in the bone marrow (BM). Activation-induced deaminase initiates isotype switching in mature B cells of secondary lymphoid structures. Previous studies questioned the strict ontological partitioning of these processes. We show that pro-B cells undergo robust switching to a subset of immunoglobulin H (IgH) isotypes. Chromatin studies reveal that in pro-B cells, the spatial organization of the Igh locus may restrict switching to this subset of isotypes. We demonstrate that in the BM, V(D)J joining and switching are interchangeably inducible, providing an explanation for the hyper-IgE phenotype of Omenn syndrome.

Histone H2AX stabilizes broken DNA strands to suppress chromosome breaks and translocations during V(D)J recombination.

The H2AX core histone variant is phosphorylated in chromatin around DNA double strand breaks (DSBs) and functions through unknown mechanisms to suppress antigen receptor locus translocations during V(D)J recombination. Formation of chromosomal coding joins and suppression of translocations involves the ataxia telangiectasia mutated and DNA-dependent protein kinase catalytic subunit serine/threonine kinases, each of which phosphorylates H2AX along cleaved antigen receptor loci. Using Abelson transformed pre-B cell lines, we find that H2AX is not required for coding join formation within chromosomal V(D)J recombination substrates. Yet we show that H2AX is phosphorylated along cleaved Igkappa DNA strands and prevents their separation in G1 phase cells and their progression into chromosome breaks and translocations after cellular proliferation. We also show that H2AX prevents chromosome breaks emanating from unrepaired RAG endonuclease-generated TCR-alpha/delta locus coding ends in primary thymocytes. Our data indicate that histone H2AX suppresses translocations during V(D)J recombination by creating chromatin modifications that stabilize disrupted antigen receptor locus DNA strands to prevent their irreversible dissociation. We propose that such H2AX-dependent mechanisms could function at additional chromosomal locations to facilitate the joining of DNA ends generated by other types of DSBs.