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. 2013 May;28(3):263-70.
doi: 10.1093/mutage/ges080. Epub 2013 Feb 5.

T-cell-specific deletion of Mof blocks their differentiation and results in genomic instability in mice

Arun Gupta  1 , Clayton R Hunt, Raj K Pandita, Juhee Pae, K Komal, Mayank Singh, Jerry W Shay, Rakesh Kumar, Kiyoshi Ariizumi, Nobuo Horikoshi, Walter N Hittelman, Chandan Guha, Thomas Ludwig, Tej K Pandita
Affiliations

T-cell-specific deletion of Mof blocks their differentiation and results in genomic instability in mice

Arun Gupta et al. Mutagenesis. 2013 May.

Abstract

Ataxia telangiectasia patients develop lymphoid malignancies of both B- and T-cell origin. Similarly, ataxia telangiectasia mutated (Atm)-deficient mice exhibit severe defects in T-cell maturation and eventually develop thymomas. The function of ATM is known to be influenced by the mammalian orthologue of the Drosophila MOF (males absent on the first) gene. Here, we report the effect of T-cell-specific ablation of the mouse Mof (Mof) gene on leucocyte trafficking and survival. Conditional Mof(Flox/Flox) (Mof (F/F)) mice expressing Cre recombinase under control of the T-cell-specific Lck proximal promoter (Mof(F/F)/Lck-Cre(+)) display a marked reduction in thymus size compared with Mof(F/F)/Lck-Cre(-) mice. In contrast, the spleen size of Mof(F/F)/Lck-Cre(+) mice was increased compared with control Mof(F/F)/Lck-Cre(-) mice. The thymus of Mof(F/F)/Lck-Cre(+) mice contained significantly reduced T cells, whereas thymic B cells were elevated. Within the T-cell population, CD4(+)CD8(+) double-positive T-cell levels were reduced, whereas the immature CD4(-)CD8(-) double-negative (DN) population was elevated. Defective T-cell differentiation is also evident as an increased DN3 (CD44(-)CD25(+)) population, the cell stage during which T-cell receptor rearrangement takes place. The differentiation defect in T cells and reduced thymus size were not rescued in a p53-deficient background. Splenic B-cell distributions were similar between Mof(F/F)/Lck-Cre(+) and Mof(F/F)/Lck-Cre(-) mice except for an elevation of the κ light-chain population, suggestive of an abnormal clonal expansion. T cells from Mof(F/F)/Lck-Cre(+) mice did not respond to phytohaemagglutinin (PHA) stimulation, whereas LPS-stimulated B cells from Mof(F/F)/Lck-Cre(+) mice demonstrated spontaneous genomic instability. Mice with T-cell-specific loss of MOF had shorter lifespans and decreased survival following irradiation than did Mof(F/F)/Lck-Cre(-) mice. These observations suggest that Mof plays a critical role in T-cell differentiation and that depletion of Mof in T cells reduces T-cell numbers and, by an undefined mechanism, induces genomic instability in B cells through bystander mechanism. As a result, these mice have a shorter lifespan and reduced survival after irradiation.

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Figures

Fig. 1.
Fig. 1.
Effect of T-cell-specific Mof inactivation on thymus and spleen size. (A) Thymus and spleen from 3-week-old Mof F/F /Lck-Cre + and Mof F/F /Lck-Cre – mice. (B) Histogram of the weight ratios of thymus and spleen to mouse body weight. The differences in thymus as well as spleen size in Mof F/F /Lck-Cre + and Mof F/F /Lck-Cre – mice are statistically significant. (C) Thymus and spleen of 12-week-old Mof F/F /Lck-Cre + and Mof F/F /Lck-Cre – mice. (D) Histogram showing the ratio of thymus and spleen to body weight. The thymus size is relatively smaller and spleen size is larger. The differences in thymus as well as spleen size in Mof F/F /Lck-Cre + and Mof F/F /Lck-Cre – mice are statistically significant. (E and F) Thymus and spleen size in p53-null background of Mof F/F /Lck-Cre + and Mof F/F /Lck-Cre – mice (E: 3 weeks old and F: 12 weeks old). *P < 0.05 and **P < 0.001 determined by the chi-square test.
Fig. 2.
Fig. 2.
Immunostaining and western blot detection of Mof expression. (A) Immunostaining of Mof in thymus and spleen of 3-week-old control mice. (B) Western blot analysis of Mof and H4K16ac levels in thymus T cells and spleen B cells from Mof F/F /Lck-Cre + and Mof F/F /Lck-Cre – mice.
Fig. 3.
Fig. 3.
Analysis of thymus and spleen lymphocytes in Mof F/F /Lck-Cre + and Mof F/F /Lck-Cre – mice. (A) Comparison of lymphocytes from (a) Mof F/F /Lck-Cre + thymus indicating a significant reduction in total lymphocytes and pan T cells (CD3+CD5+) but increased pan B cells (B220+) levels; (b) thymic B cells from Mof F/F /Lck-Cre + mice indicate a significant increase in mature B cells (IgM+IgD+) with an unusually high clonal (κ light-chain) expansion ratio (κ); and (c) thymic T cells from Mof F/F /Lck-Cre + indicating an early developmental defect, increased population of CD4–CD8– (DN) T cells as well as statistically significant decreased CD4+CD8+ T cells. DN population was further classified into DN1 (CD44+CD25–), DN2 (CD44+CD25+), DN3 (CD44–CD25+) and DN4 (CD44–CD25–) populations and T cells from Mof F/F /Lck-Cre + accumulated in DN3 and DN4. (B) Comparison of cells from spleen (a) statistically significant reduction in total lymphocytes and T cells, whereas total B-cell number was unchanged; (b) difference in B cells, mature B cell (IgM+IgD+) was unchanged; however, abnormal κ light-chain clonal expansion was observed; (c) difference in splenic T cells, the number of helper T cells (CD4+CD8–) was unchanged; however, cytotoxic T-cells (CD4–CD8+) level was reduced. *P < 0.05 and **P < 0.001 determined by the chi-square test.
Fig. 4.
Fig. 4.
Effect of p53-null status on thymic and splenic cell populations in Mof F/F /Lck-Cre + mice. (A) Comparison of cells from thymus (a) B cells and (b) T cells present no significant differences in cell types between p53-null and wild-type background of Mof F/F /Lck-Cre + mice; however, significant differences were observed between Mof F/F /Lck-Cre + and Mof F/F /Lck-Cre – mice as described in Figure 3. (B) Comparison of cells from spleen. (a) B cells and (b) T cells present no significant differences in cell types of p53-null and wild-type background Mof F/F /Lck-Cre + mice; however, some differences were observed between Mof F/F /Lck-Cre + and Mof F/F /Lck-Cre – mice as described in Figure 3. *P < 0.05 and **P < 0.001 determined by the chi-square test.
Fig. 5.
Fig. 5.
Genomic instability of T cells and B cells in Mof F/F /Lck-Cre + mice. (A) T cells from thymus of 3-week-old mice showing nuclei of different sizes. Telomere signals (green) are detected by using telomere-specific probe. DNA was stained with DAPI (4′,6-diamidino-2-phenylindole) (blue). (B) T cells from thymus of 12-week-old mice. (B-1) DAPI staining and (B-2) telomere signals detected by FISH. Red arrows indicate the fragmented nuclei with or without telomere signals. (C) Metaphases from B cells of 3-week-old mice (C-1) DAPI staining and (C-2) telomere FISH staining, red arrows showing loss of telomere signals and white arrow showing a fragment with telomere signal. (D) Metaphase from 6-week-old mouse B cells showing chromosome end-to-end associations (yellow arrow) and telomere fusion leading to Robertsonian mutation (white arrow). (E) Giemsa staining of metaphase from 12-week-old mouse B cells showing chromosome fragments. (F) Metaphase of B cells showing fragment with telomere signal and (G) metaphase of B cells with loss or reduced telomere signal (red arrows) and high frequency of telomere fusions (white arrows). (H) Metaphase segment from 15-week-old mouse B cells showing telomere fusions leading to Robertsonian mutations (H-1) DAPI staining, white arrows showing chromosome end fusions and (H-2) telomere FISH showing loss of telomere signal (red arrows) and telomere fusions without any telomere signals (white arrows).
Fig. 6.
Fig. 6.
Effect of T-cell-specific Mof depletion on weight and post-irradiation survival. (A) Body weight and (B) survival after 3 Gy IR exposure of Mof F/F /Lck-Cre + and Mof F/F /Lck-Cre – mice. The differences in the weight and survival of Mof F/F /Lck-Cre + and Mof F/F /Lck-Cre – mice are modest, but statistically significant (P < 0.05 determined by the chi-square test). The cumulative survival was plotted according to Kaplan–Meier analysis.
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