Disruption of pre-TCR expression accelerates lymphomagenesis in E2A-deficient mice

Abstract

The helix-loop-helix proteins E47 and E12, which are encoded by the E2A gene, regulate several stages of T cell development. In addition, mice deficient for E2A are highly susceptible to thymic lymphoma. Here we report that the development of lymphoma in E2A-deficient mice did not require pre- and recombinase-activating gene expression. Rather, we found that, whereas illegitimate DNA rearrangement did not play a major role in the development of these lymphomas, defects that prevented pre-T cell antigen receptor expression tended to accelerate lymphomagenesis in E2A-deficient mice. These data and previous observations also provide insight into the role of Notch in lymphoma development. Specifically, we propose that Notch activation indirectly modulates E2A activity through induction of pre-Talpha expression, ultimately leading to the development of lymphoma.

Fig 1.

Morbidity from thymic lymphoma in E47-null mice is accelerated by homozygosity for the scid mutation. (A) Plot comparing the rate of lymphoma morbidity in E47−/− mice with scid/+ and scid/scid genotypes. (B) Plot of lymphoma morbidity in scid/scid mice with E47+/− and E47+/+ genotypes. The age at which mice were killed or died of lymphoma is plotted against the fraction of mice that remained outwardly healthy. The number of mice of each genotype is indicated in parentheses in the symbol legend. The data represent those mice that either were shown to have become ill or died because of lymphoma or survived for at least 1 year without becoming ill. Two E47−/− scid/scid, two E47−/− scid/+, four E47+/− scid/scid, and four E47+/+ scid/scid mice were found dead and were unable to be necropsied, whereas three E47+/− scid/scid and one E47 wild-type scid/scid mice died or were euthanized because of illnesses other than lymphoma; these mice were excluded from the data presented.

Fig 2.

Morbidity from thymic lymphoma in E47-null mice is accelerated by homozygosity for the TCRβ mutation. The age at which mice were killed or died of lymphoma is plotted against the fraction of mice that remained outwardly healthy. The number of mice of each genotype is indicated in parentheses in the symbol legend. The data represent those mice that either were shown to have become ill or died because of lymphoma or survived for at least 1 year without becoming ill. Three E47−/− TCRβ+/− and five E47−/− TCRβ−/− that were found dead and not subjected to necropsy were excluded from the data presented.

Fig 3.

Effect of RAG-1 deficiency on morbidity from thymic lymphoma in E47-null mice. (A) Plot comparing the rate of lymphoma morbidity in E47−/− mice with RAG-1+/+, +/−, and −/− genotypes. (B) Plot comparing the rate of lymphoma morbidity in E47+/− mice with RAG-1−/− and RAG-1+/− genotypes. The age at which mice were killed or died of lymphoma is plotted against the fraction of mice that remained outwardly healthy. The number of mice of each genotype is indicated in parentheses in the symbol legend. The data represent those mice that either were shown to have become ill or died because of lymphoma or survived for at least 1 year without becoming ill. Five E47−/− RAG-1+/−, two E47−/− RAG-1−/−, and ten E47+/− RAG-1−/− were found dead and were not subjected to necropsy, whereas one E47−/− RAG-1+/− and three E47+/− RAG-1−/− mice were found not to have thymic lymphoma upon necropsy; these mice were excluded from the data presented. (C) Thymic lymphomas from E47+/− RAG-1−/− mice express E47. Immunoblot with nuclear extracts from two E47+/− RAG-1−/− lymphomas (lanes 1–2), whole cell extract from a third E47+/− RAG-1−/− lymphoma, nuclear extract from a E47−/− scid/scid lymphoma (lane 4), and whole-cell extract from the p53−/− T lymphoma line 16610D9 (49) (lane 5) was probed with the anti-E47 antibody G127–32 (PharMingen).

Fig 4.

Phenotypic analysis of lymphomas from E47-deficient mice with defects in TCRβ expression. Lymphomas from mice with E47−/− scid/scid, E47+/− RAG-1−/−, E47−/− RAG-1−/−, E47−/− TCRβ−/−, and E47−/− TCRβ+/− genotypes were analyzed by flow cytometry for surface expression of CD25, CD44, CD8, and CD4 as well as for DNA content. Each column of plots represents the analysis of one lymphoma with the genotype indicated at the top. (Top) Dot plots depicting surface CD25 and CD44 expression; (Middle) Dot plots indicating surface CD8 and CD4 levels. (Bottom) Histograms showing the DNA content measured by using propidium iodide, with the percentage of cells with greater than 2N listed in the top right corner of each histogram. The DNA content histogram from wild-type thymocytes is also provided for comparison.

Fig 5.

Model depicting an indirect pathway linking expression of activated Notch (NotchIC) with the inhibition of E2A. NotchIC has been shown to activate the pre-Tα promoter through association with the CSL transcription factor, and thus may induce aberrantly high levels of the pre-TCR complex (48). Signaling through the antigen receptor complex in thymocytes acts to inhibit E2A activity, at least in part through the induction of Id3 transcription by a Ras/mitogen-activated protein kinase (Ras/MAPK)-dependent pathway that activates the early growth response gene 1 (Egr-1) (23).