Faculty Bibliography

Scientists are by nature a determined, dedicated breed and do not give up easily even when confronted with seemingly insurmountable obstacles. So why would anyone who has embarked along this career path choose to take a break from it, and can one really return to a successful academic career after this break?

Deregulated activation of beta-catenin in cancer has been correlated with genomic instability. During thymocyte development, beta-catenin activates transcription in partnership with T-cell-specific transcription factor 1 (Tcf-1). We previously reported that targeted activation of beta-catenin in thymocytes (CAT mice) induces lymphomas that depend on recombination activating gene (RAG) and myelocytomatosis oncogene (Myc) activities. Here we show that these lymphomas have recurring Tcra/Myc translocations that resulted from illegitimate RAG recombination events and resembled oncogenic translocations previously described in human T-ALL. We therefore used the CAT animal model to obtain mechanistic insights into the transformation process. ChIP-seq analysis uncovered a link between Tcf-1 and RAG2 showing that the two proteins shared binding sites marked by trimethylated histone-3 lysine-4 (H3K4me3) throughout the genome, including near the translocation sites. Pretransformed CAT thymocytes had increased DNA damage at the translocating loci and showed altered repair of RAG-induced DNA double strand breaks. These cells were able to survive despite DNA damage because activated beta-catenin promoted an antiapoptosis gene expression profile. Thus, activated beta-catenin promotes genomic instability that leads to T-cell lymphomas as a consequence of altered double strand break repair and increased survival of thymocytes with damaged DNA.

It is nearly 30 years since the Alt lab first put forward the accessibility model, which proposes that cleavage of the various antigen receptor loci is controlled by lineage and stage specific factors that regulate RAG access. Numerous labs have since demonstrated that locus opening is regulated at multiple levels that include sterile transcription, changes in chromatin packaging, and alterations in locus conformation. Here we focus on the interplay between transcription and RAG binding in facilitating targeted cleavage. We discuss the results of recent studies that implicate transcription in regulating nuclear organization and altering the composition of resident nucleosomes to promote regional access to the recombinase machinery. Additionally we include new data that provide insight into the role of the RAG proteins in defining nuclear organization in recombining T cells.

Translocations occur through the aberrant joining of large stretches of non-contiguous chromosomal regions. The substrates for these illegitimate rearrangements can arise as a result of damage incurred during normal cellular processes, such as transcription and replication, or through the action of genotoxic agents. In lymphocytes many translocations bear signs of having originated from abnormalities introduced during programmed recombination. Although recombination is tightly controlled at different levels, mistakes can occur leading to cytogenetic anomalies that include deletions, insertions, amplifications and translocations, which are an underlying cause of leukemias and lymphomas. In this review we focus on recent studies that provide insight into the origins of translocations that arise during the two lymphocyte specific programmed recombination events: V(D)J and class switch recombination (CSR).

V(D)J recombination is essential for generating a diverse array of B and T cell receptors that can recognize and combat foreign antigens. As with any recombination event, tight control is essential to prevent the occurrence of genetic anomalies that drive cellular transformation. One important aspect of regulation is directed targeting of the RAG recombinase. Indeed, RAG accumulates at the 3' end of individual antigen receptor loci poised for rearrangement; however, it is not known whether focal binding is involved in regulating cleavage, and what mechanisms lead to enrichment of RAG in this region. Here, we show that monoallelic looping out of the 3' end of the T cell receptor alpha (Tcra) locus, coupled with transcription and increased chromatin/nuclear accessibility, is linked to focal RAG binding and ATM-mediated regulation of monoallelic cleavage on looped-out 3' regions. Our data identify higher-order loop formation as a key determinant of directed RAG targeting and the maintenance of genome stability.