Aberrant V(D)J recombination is known to contribute to development of lymphoid neoplasias and has been associated with some nonlymphoid cancers and tumor cell lines. The long-term objective of this project is to analyze age and tissue-specific constraints on accessibility to aberrant V(D)J recombination in vivo. We will determine the frequency of aberrant and ectopic RAG-mediated DNA rearrangements in a variety of tissues of RAG transgenic animals (and WT controls) induced during different windows of development including fetus, neonate and adult.
The specific aims of the project will focus on detection of functional RAG protein expression in different tissues of inducible RAG transgenic mice, and examination of tissues expressing RAG for accessibility to aberrant V(D)J-related DNA rearrangements and strand breaks. We will examine recently-described """"""""hotspots"""""""" for aberrant V(D)J rearrangements involving """"""""cryptic"""""""" V(D)J recombination signal sequences (RSSs), and """"""""ectopic"""""""" immunoglobulin (Ig) and T cell receptor (TCR) loci rearrangements usually only associated with developing lymphocytes, but found previously in nonlymphoid cell lines with forced RAG expression. The following specific aims will be addressed:
Specific aim 1 will measure transgenic RAG mRNA expression in a variety of tissues in induced RAG transgenic animals using reverse transcription-dependent PCR. Functional transgenic RAG protein activity in different tissues in animals induced at different ages will be confirmed by breeding RAG transgenic animals to a V(D)J reporter substrate transgenic strain. In induced mice harboring RAG transgenes and reporter substrate transgene, chromosomally integrated substrate recombination will be detected by PCR.
Specific aim 2 will measure the frequency of aberrant RAG-mediated V(D)J events including a TCR D21 RSS reinsertion event on Chromosome 1, and deletions in the Notch1 oncogene and Rit1/Bcl11b tumor suppressor gene, all observed recently with measurable frequencies by direct PCR in thymus of normal mice. We will compare the availability for rearrangements (or DNA breaks that precede them) in thymus at different stages of normal animal development, and in thymus and nonlymphoid tissues of RAG-deficient animals subjected to RAG transgene induction at different developmental windows.
Specific aim 3 will measure the ability of Ig/TCR loci previously shown to rearrange with forced RAG expressed in embryonic kidney cell lines to rearrange in a variety of nonlymphoid cells in vivo.
This aim will address the hypothesis that embryonic nonlymphoid cells are more accessible to ectopic RAG-mediated recombination at these loci than those of the adult. This project will provide novel information on accessibility to RAG-mediated aberrant and ectopic V(D)J recombination events in a variety of mouse tissues and will enhance our understanding of the relationship between RAG proteins and development of neoplasias.
The Recombination Activating Gene (RAG) proteins mediate normal gene rearrangements in developing lymphocytes, but aberrant rearrangements can contribute to development of a variety of lymphocyte tumors. A role for these proteins in the development of other types of tumors has been proposed, but more studies are needed to understand if RAGs cause detrimental gene rearrangements in nonlymphoid cells. Analysis of aberrant and ectopic gene rearrangements in a variety of tissues of inducible RAG transgenic mice at different developmental stages will further our understanding of the potential for the RAG proteins to contribute to cancer development both in lymphoid and nonlymphoid cells.
| Champagne, Devin P; Shockett, Penny E (2014) Illegitimate V(D)J recombination-mediated deletions in Notch1 and Bcl11b are not sufficient for extensive clonal expansion and show minimal age or sex bias in frequency or junctional processing. Mutat Res 761:34-48 |
