Tight control of antigen-receptor gene rearrangement is required to preserve genome integrity and prevent the occurrence of damage and translocations that lead to leukemia and lymphoma. Our most recent efforts have focused on identifying the mechanism underlying regulation of RAG cleavage in individual cells. These studies have revealed that higher-order looping and nuclear organization of antigen receptor loci facilitates regulated, coordinated rearrangement in recombination centers. In brief, we discovered that the formation of large higher-order mono-allelic RAG-dependent loops, which separate the 3' end of Tcra from its chromosome territory, correlates with targeting of RAG binding and mono-allelic cleavage in this region. RAG- dependent higher-order looping facilitates RAG-dependent association of homologous (Tcra) and heterologous (Tcra/Igh) antigen receptor alleles at the time of recombination. Moreover, mono-allelic, mono- locus cleavage and the maintenance of genome stability are linked to ATM-mediated regulation of higher- order mono-allelic, mono-locus loop formation and nuclear organization of Tcra and Igh. These data support a model for feedback control of RAG activity occurring in localized recombination centers. In this renewal we aim to expand on our previous findings to further test our model and explore the mechanisms underlying ATM and RAG-mediated control of cleavage. Higher-order looping out of Tcra from the chromosome territory defines a form of looping in recombination that is distinct from the well-known intra-locus looping / contraction tha we and others previously discovered. Thus, our finding adds an entirely new layer of regulation to the rearrangement process. We will examine the relationship between structure and function by performing a detailed analysis of the Tcra/d locus at different stages of development in the presence and absence of RAG. Our approach will be to combine novel experimental and theoretic simulation using high-throughput automated analyses with highest resolution FISH (HTHR-FISH), and high-throughput monitoring of intra/inter chromosomal contacts by chromosome conformation capture (4C-seq). To determine how higher-order looping is integrated with (i) focal RAG binding, (ii) chromatin modifications, (iii) RNA levels and (iv) nuclear accessibility to control RAG cleavage in the interest of preserving genomic stability we will examine the effects of different repair proteins (ATM, 53BP1 and Artemis) in regulating chromosome dynamics of Tcra/d, Igh, Tcrb, and Tcrg (pairing, higher order looping and repositioning to repressive pericentromeric heterochromatin) to control breaks and damage on these loci. This will provide insight into how altered regulation, in the absence of individual repir factors, impacts on the translocation signature of mutant cells. Finally, we aim to determine whether ATM-mediated negative feedback regulation of RAG cleavage involves inhibition of RAG enzymatic activity.
We have been investigating the regulation of ordered, sequential recombination of antigen receptor loci. Our recent studies have brought to light new regulatory aspects of rearrangement that involve ATM and RAG mediated control of higher-order mono-allelic, mono-locus looping, which is linked to mono-allelic, mono-locus recombination. In this renewal we aim to test our hypothesis that feedback control of RAG activity in localized recombination centers maintains genome stability during recombination through changes in nuclear organization that depend on cooperation between RAG and ATM.
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