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

Abstract

Illegitimate V(D)J recombination at oncogenes and tumor suppressor genes is implicated in formation of several T cell malignancies. Notch1 and Bcl11b, genes involved in developing T cell specification, selection, proliferation, and survival, were previously shown to contain hotspots for deletional illegitimate V(D)J recombination associated with radiation-induced thymic lymphoma. Interestingly, these deletions were also observed in wild-type animals. In this study, we conducted frequency, clonality, and junctional processing analyses of Notch1 and Bcl11b deletions during mouse development and compared results to published analyses of authentic V(D)J rearrangements at the T cell receptor beta (TCRβ) locus and illegitimate V(D)J deletions observed at the human, nonimmune HPRT1 locus not involved in T cell malignancies. We detect deletions in Notch1 and Bcl11b in thymic and splenic T cell populations, consistent with cells bearing deletions in the circulating lymphocyte pool. Deletions in thymus can occur in utero, increase in frequency between fetal and postnatal stages, are detected at all ages examined between fetal and 7 months, exhibit only limited clonality (contrasting with previous results in radiation-sensitive mouse strains), and consistent with previous reports are more frequent in Bcl11b, partially explained by relatively high Recombination Signal Information Content (RIC) scores. Deletion junctions in Bcl11b exhibit greater germline nucleotide loss, while in Notch1 palindromic (P) nucleotides are more abundant, although average P nucleotide length is similar for both genes and consistent with results at the TCRβ locus. Non-templated (N) nucleotide insertions appear to increase between fetal and postnatal stages for Notch1, consistent with normal terminal deoxynucleotidyl transferase (TdT) activity; however, neonatal Bcl11b junctions contain elevated levels of N insertions. Finally, contrasting with results at the HPRT1 locus, we find no obvious age or gender bias in junctional processing, and inverted repeats at recessed coding ends (Pr nucleotides) correspond mostly to single-base additions consistent with normal TdT activity.

Genetic Analysis of T Cell Lymphomas in Carbon Ion-Irradiated Mice Reveals Frequent Interstitial Chromosome Deletions: Implications for Second Cancer Induction in Normal Tissues during Carbon Ion Radiotherapy

“…Clonal expansion of cells with LOH at the Bcl11b locus has been reported as being one of the earliest detectable changes (along with Myc amplification) in the thymus post-irradiation but prior to the onset of lymphoma [ 37 ]. The presence of Notch1 Type 1 deletion-bearing cells in the normal mouse thymus [ 38 ] suggests that activating Notch1 deletions in the TL could result from selective expansion of cells with pre-existing spontaneous deletions, yet other evidence indicates that the recombination-mediated deletions are a later spontaneous event in the carcinogenic sequence [ 37 , 39 ], with no increase in deletion frequency in TL from irradiated compared to unirradiated mice [ 31 ]. Since studies of large panels of T-ALL have failed to find equivalent recombination-mediated deletions in the human NOTCH1 gene [ 32 ], this represents a mouse-specific tumour susceptibility that may contribute to their increased vulnerability to radiation-induced T cell tumours; though, the finding of a NOTCH1 Type 2 activating deletion (not recombination mediated) in a T-ALL patient [ 40 ] demonstrates that the mechanism has prima facie relevance to human cancers.…”

iRAGu: A Novel Inducible and Reversible Mouse Model for Ubiquitous Recombinase Activity

“…We then observed that Bcl11b cryptic rearrangements were also detectable in two Tg endoR2 −/− animals induced by TAM, albeit approximately 10 times less frequently than in WT [(33, 37); Figure 4B; Table 2]. Such cryptic rearrangements were not detected in the 1 million cells equivalent DNA tested for the third Tg endoR2 −/− TAM + animal (Figure 4).…”

“…We used fluctuation PCR of total thymocytes DNA to assess frequencies of Bcl11b recombination which was similar in WT (25 rearrangements per million thymocytes) and RAG-competent Tg animals (27.7 rearrangements per million thymocytes), and in the range of frequencies documented previously [23–50 rearrangements per million thymocytes in Ref. (37) and Ref. (33), respectively; data not shown].…”