Notch and the pre-TCR coordinate thymocyte proliferation by induction of the SCF subunits Fbxl1 and Fbxl12

Abstract

Proliferation is tightly regulated during T cell development, and is limited to immature CD4CD8 thymocytes. The major proliferative event is initiated at the ‘β-selection’ stage following successful rearrangement of Tcrβ, and is triggered by and dependent on concurrent signaling by Notch and the pre-T cell receptor (TCR); however, it is unclear how these signals cooperate to promote cell proliferation. Here, we found that β-selection-associated proliferation required the combined activity of two Skp-cullin-F-box (SCF) ubiquitin ligase complexes that included as substrate recognition subunits the F-box proteins Fbxl1 or Fbxl12. Both SCF complexes targeted the cyclin-dependent kinase inhibitor Cdkn1b for polyubiquitination and proteasomal degradation. We found that Notch signals induced the transcription of Fbxl1, whereas pre-TCR signals induced the transcription of Fbxl12. Thus, concurrent Notch and pre-TCR signaling induced the expression of two genes, Fbxl1 and Fbxl12, whose products functioned identically but additively to promote degradation of Cdkn1b, cell cycle progression, and proliferation of β-selected thymocytes.

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Fig. 1: SCF-Fbxl12 complexes target Cdkn1b for polyubiquitination and proteasomal degradation.
Fig. 2: Impaired β-selection-associated proliferation in Lck-Cre Fbxl12fl/fl mice.
Fig. 3: Restoration of thymocyte development and β-selection-associated proliferation in Lck-Cre Fbx12fl/fl mice by deletion of Cdkn1b.
Fig. 4: Thymocyte development and β-selection-associated proliferation are strongly impaired in thymocytes lacking Fbxl1 and Fbxl12.
Fig. 5: Fbxl1 and Fbxl12 target the same site on Cdkn1b for K48 polyubiquitination.
Fig. 6: Notch signaling and pre-TCR signaling regulate Fbxl1 and Fbxl12 expression, respectively.
Fig. 7: Fbxl1 and Fbxl12 function interchangeably to promote proliferation, but are not sufficient for β selection.
Fig. 8: Proliferation of immature γδTCR+ thymocytes is mediated primarily by TCR-induced regulation of Fbxl12.

Data availability

The data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

This work was supported by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (project number 1ZIAHD001803-25 to P.E.L.), a grant from the Swedish Society for Medical Research (to B.Z.), the Canadian Institutes of Health Research (FND-154332 to J.C.Z.-P.) and the National Institutes of Health (1P01AI102853-01 to J.C.Z.-P.). J.C.Z.-P. is supported by a Canada Research Chair in Developmental Immunology, and K.Y. is supported by a Canada Graduate Scholarship from the Natural Sciences and Engineering Research Council of Canada. The authors thank R. Bosselut for critical reading of the manuscript, and R. Bosselut, A. Bhandoola, B. J. Fowlkes, N. Taylor and H. Petrie for helpful discussions on the project.

Author information

B.Z., K.Y., L.L. and J.Y.L. performed the experiments. B.Z., P.E.L. and J.C.Z.-P. designed the experiments. P.E.L. wrote the manuscript.

Correspondence to Paul E. Love.

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Integrated supplementary information

Supplementary Figure 1 β-selection-associated proliferation is increased in Lck-Cre Cdkn1bfl/fl mice and decreased in Fbxl1-/- mice, and deletion of Cdkn1b rescues thymocyte development in Fbxl1-/- mice.

(a) Flow cytometry analysis showing the phenotype of thymocytes (left) or splenocytes (right) from mice of the indicated genotype. Thymus: left, CD4 vs CD8 staining of total thymocytes; center, CD44 vs CD25 staining of lineage-negative DN thymocytes. Spleen: CD4 vs CD8 staining of total splenocytes; (b) Immunoblot analysis showing Cdkn1b protein expression in purified lineage-negative DN thymocytes from Lck-Cre or Lck-Cre Cdkn1bfl/fl (Cdkn1b KO) mice. (c) Cell numbers of total thymocytes or the indicated thymocyte subsets and DN3/DN4 ratio (n=4 mice per genotype). (d) Numbers of CD4 SP and CD8 SP cells in the thymus and spleen of indicated genotype (n=4 mice per genotype). For all graphs, horizontal lines indicate the mean and vertical lines indicate the standard deviation (±s.d.), P values were determined by unpaired two-tailed Student’s t-test. NS, not significant (P>0.05), *P<0.05, **P<0.01, ***P<0.005, ****P<0.0001. Data shown in (a) and (b) are representative of four or two independent experiments, respectively.

Supplementary Figure 2 β-selection-associated proliferation is increased in Lck-Cre Cdkn1bfl/fl mice and decreased in Fbxl1-/- mice and Fbxl1 is a subunit of an SCF ubiquitin ligase complex that targets Cdkn1b.

(a) Cell cycle analysis of thymocyte subsets showing the percentage of cells in S/G2/M phase. (n=4 mice per genotype). (b) Percentage of annexin V positive cells within the indicated thymocyte subsets from Fbxl1+/+ or Fbxl1-/- mice. (n=3 mice per genotype). (c) Immunoprecipitation and immunoblot analysis showing the interaction of Fbxl1 and Cul1 in HEK-293T cells transfected with plasmids encoding Myc-Fbxl1 and Flag-Cul1. (d) Immunoprecipitation and immunoblot analysis showing the interaction of Fbxl1 and Cdkn1b in HEK-293T cells transfected with plasmids encoding Myc-Fbxl1 and Flag-Cdkn1b. (e) Immunoblot analysis showing degradation of Cdkn1b by SCF-Fbxl1 complexes and its dependence on the Fbxl1 F-box motif in HEK-293T cells transfected with plasmids encoding Myc-Fbxl1 or Myc-Fbxl1ΔF (Myc epitope tagged Fbxl1 lacking the F-box motif) and Flag-Cdkn1b. (f) Immunoblot analysis showing Fbxl1 and Cdkn1b protein expression in purified DN thymocytes from Fbxl1+/+ or Fbxl1-/- mice. For all graphs, small horizontal lines indicate the mean and vertical lines indicate the standard deviation (±s.d.), P values were determined by unpaired two-tailed Student’s t-test. NS, not significant (P>0.05), *P<0.05, **P<0.01, ***P<0.005, ****P<0.0001. Data shown in (c-f) are representative of three independent experiments.

Supplementary Figure 3 Analysis of Fbxl12 expression in human and mouse tissues and generation of Fbxl12fl/fl mice.

(a) BioGPS database plot showing expression of Fbxl12 mRNA in human tissues evaluated by microarray. (http://www.biogps.org). (b) Immgen plot showing expression of Fbxl12 mRNA in mouse tissues and thymocyte subsets detected by microarray. (http://www.immgen.org). (c) Immunoblot analysis showing Fbxl12 and Fbxl1 expression in wild type (B6) thymocyte subsets. (d) Schematic showing gene targeting scheme for generation of Fbxl12 conditional knockout mice. (e) PCR analysis of genomic DNA purified from the tail snips of wild-type (+/+), heterozygous (fl/+) and homozygous(fl/fl) Fbxl12 floxed mice showing WT and Floxed Fbxl12 alleles. (f) Immunoblot analysis showing Fbxl12, Cdkn1b, and β-actin in total thymocytes from Lck-Cre, and Lck-Cre Fbxl12fl/fl mice. Data shown in (c,e,f) are representative of 3 independent experiments.

Supplementary Figure 4 Impaired β-selection-associated cell cycling and proliferation in Lck-Cre Fbxl12fl/fl mice.

(a) Cell cycle analysis of thymocyte subsets from mice of the indicated genotype showing percentage of S/G2/M in the indicated thymocyte subsets. (b) Numbers of CD4 SP and CD8 SP cells in the thymus and spleen of mice of the indicated genotype (n=6 mice per genotype). (c) Percentage of annexin V positive cells within thymocyte subsets from mice of the indicated genotype (n=3 mice per genotype). For all graphs horizontal lines indicate the mean and vertical lines indicate standard deviation (±s.d.), P values were determined by unpaired two-tailed Student’s t-test. NS, not significant (P>0.05), *P<0.05, **P<0.01, ***P<0.005, ****P<0.0001. Data shown in (a) are one representative of five experiments.

Supplementary Figure 5 Analysis of RORγt expression in Lck-Cre Fbxl12fl/fl mice and phenotype of AND TCR transgenic (tg) Lck-Cre Fbxl12fl/fl mice.

(a) Immunoblot analysis showing RORγt and β-actin loading control in thymocytes of the indicated genotype. (b) Flow cytometry analysis of thymocytes from AND TCR transgenic (tg) Fbxl12fl/fl and Lck-Cre Fbxl12fl/fl mice. Shown are: left, CD4 vs CD8 staining of total thymocytes; center, histogram of Vα11 staining of total thymocytes; right, CD44 vs CD25 staining of DN thymocytes. (c) Total thymocyte numbers and ratio of DN3 stage cells to DN4 stage cells (n=4 mice per genotype) in AND-tg Fbxl12fl/fl and AND-tg Lck-Cre Fbxl12fl/fl mice. For all graphs, horizontal lines indicate the mean and vertical lines indicate standard deviation (±s.d.), P values were determined by unpaired two-tailed Student’s t-test. NS, not significant (P>0.05), *P<0.05, **P<0.01, ***P<0.005, ****P<0.0001. Data shown in (a) and (b) are one representative of three experiments.

Supplementary Figure 6 β-selection-associated proliferation is strongly impaired in the absence of Fbxl1 and Fbxl12.

(a) Cell cycle analysis of thymocyte subsets from mice of the indicated genotype showing percentage of S/G2/M cells. (b) Numbers of CD4 SP and CD8 SP cells in the thymus and spleen of mice of the indicated genotype (n=5 mice per genotype). (c) Percentage of annexin V positive cells within the thymocyte subsets of the indicated genotype (n=3 mice per genotype). For all graphs, horizontal lines indicate the mean and vertical lines indicate standard deviation (±s.d.), P values were determined by unpaired two-tailed Student’s t-test. NS, not significant (P>0.05), *P<0.05, **P<0.01, ***P<0.005, ****P<0.0001. Data shown in (a) are representative of four independent experiments.

Supplementary Figure 7 β-selection associated proliferation is impaired in Fbxl1+/-Fbxl12+/- compound heterozygous thymocytes.

(a) Flow cytometry of cells from Thymus (left) or Spleen (right) from mice of the indicated genotype. Thymus: CD4 vs CD8 staining of total thymocytes; center, CD44 vs CD25 staining of lineage-negative DN thymocytes. Spleen: CD4 vs CD8 staining of total splenocytes. (b) Immunoblot analysis showing Fbxl1, Fbxl12, Cdkn1b and β-actin in purified lineage-negative DN thymocytes from mice of the indicated genotype. (c) Cell numbers and DN3/DN4 ratio of thymocytes from mice of the indicated genotype (n=4 mice per genotype). (d) Percentage of cycling S/G2/M stage cells in the indicated thymocyte subsets (n=4 mice per genotype). For all graphs, horizontal lines indicate the mean and vertical lines indicate standard deviation (±s.d.), P values were determined by unpaired two-tailed Student’s t-test. NS, not significant (P>0.05), *P<0.05, **P<0.01, ***P<0.005, ****P<0.0001. Data shown in (a) and (b) are representative of three or two independent experiments, respectively.

Supplementary Figure 8 Time-course of Fbxl1 and Fbxl12 induction and cell cycle analysis of thymocyte subsets.

(a) Real-time PCR analysis showing Fbxl1 and Fbxl12 mRNA expression in Rag2-/- thymocytes plated on OP9-DL1 cells for the indicated times. mRNA expression is relative to Day 0. Data shown are combined results of three experiments. (b) Real-time PCR analysis showing Fbxl1 and Fbxl12 mRNA expression in thymocytes of Rag2-/- mice that were injected (IP) with anti-CD3 antibody and harvested at the indicated times. mRNA expression is relative to Day 0. Data shown are combined results of three experiments. (c) Cell cycle analysis of thymocyte subsets from mice of the indicated genotype in Fig. 7a showing percentage of cells in S/G2/M phase. For all graphs, horizontal lines indicate the mean and vertical lines indicate the standard deviation (±s.d.), P values were determined by unpaired two-tailed Student’s t-test. NS, not significant (P>0.05), *P<0.05, **P<0.01, ***P<0.005, ****P<0.0001. Data shown in (c) are representative of three independent experiments.

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Zhao, B., Yoganathan, K., Li, L. et al. Notch and the pre-TCR coordinate thymocyte proliferation by induction of the SCF subunits Fbxl1 and Fbxl12. Nat Immunol 20, 1381–1392 (2019). https://doi.org/10.1038/s41590-019-0469-z

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