Ellen Rothman Richie

Table S1 shows predicted off target sites for p53TD mutant mice. Table S2 shows upregulated PPAR target genes in p53AD vs p53-null. Table S3 shows ATAC-seq and RNA-seq congruent genes in p53AD vs p53 null. Table S4 shows p53half site analysis in PPAR target genes Table S5 shows control sequences used for p53 half site analysis Table S6 shows altered genes in p53AD-derived thymic lymphomas Table S7 shows primers used for genotyping of p53 mutant mice Table S8 shows primers used for qRT-PCR Table S9 shows antibodies used for flow cytometry Figure S1 shows extended data for generation of knock-in mouse models and features of p53TD mutant proteins and mice. Figure S2 shows homozygous mutant mice survival curves as well as tumor data. Figure S3 shows exome sequencing results for thymic lymphomas and hemangiosarcomas derived from p53 monomer and dimer mutant mice. Figure S4 shows characterization of stress responses of p53TD mutants. Figure S5 shows RNA-sequencing results for p53AD and p53RP, and Mdm2 genetic deletion rescue experiments. Figure S6 shows RNA-seq results of basal p53AD vs p53-null thymi, upstream regulator analysis, and ATAC-seq results for specific loci. Figure S7 shows extended data for p53 dimer mutant novel activities (co-IP, motif analysis, other dimer mutants activities, and tumor data) Figure S8 shows ferroptosis and scRNA-seq extended data Figure S9 shows diagram summarizing findings within this study.

SUMMARY: T cells in the perinatal thymus have distinct phenotypes and functions that may be instructed by age-specific features of the microenvironment. We evaluated molecular and cellular profiles of thymic stromal cells, including thymic epithelial cells (TECs), mesenchyme, endothelium, and hematopoietic antigen presenting cells (hAPCs), from birth through one-month of age in mice. Single-cell transcriptional profiling, flow cytometry, and immunohistochemistry revealed coordinated stromal changes accompanied by altered thymocyte differentiation at defined transitional ages during the shift from perinatal growth to juvenile homeostasis, which was mirrored in humans. These analyses link diminished IGF2 expression by mesenchymal cells with activation of the RB pathway in TECs at the transition. Moreover, a coordinated increase in type I interferon signaling in stroma across the transition is associated with altered antigen processing and presentation signatures in TECs and hAPCs. Collectively, these datasets provide a resource to interrogate thymic stroma across the perinatal to juvenile transition.

Age‐related thymus involution results in decreased T‐cell production, contributing to increased susceptibility to pathogens and reduced vaccine responsiveness. Elucidating mechanisms underlying thymus involution will inform strategies to restore thymopoiesis with age. The thymus is colonized by circulating bone marrow (BM)‐derived thymus seeding progenitors (TSPs) that differentiate into early T‐cell progenitors (ETPs). We find that ETP cellularity declines as early as 3 months (3MO) of age in mice. This initial ETP reduction could reflect changes in thymic stromal niches and/or pre‐thymic progenitors. Using a multicongenic progenitor transfer approach, we demonstrate that the number of functional TSP/ETP niches does not diminish with age. Instead, the number of pre‐thymic lymphoid progenitors in the BM and blood is substantially reduced by 3MO, although their intrinsic ability to seed and differentiate in the thymus is maintained. Additionally, Notch signaling in BM lymphoid progenitors and in ETPs diminishes by 3MO, suggesting reduced niche quality in the BM and thymus contribute to the early decline in ETPs. Together, these findings indicate that diminished BM lymphopoiesis and thymic stromal support contribute to an initial reduction in ETPs in young adulthood, setting the stage for progressive age‐associated thymus involution.

Table S1 shows predicted off target sites for p53TD mutant mice. Table S2 shows upregulated PPAR target genes in p53AD vs p53-null. Table S3 shows ATAC-seq and RNA-seq congruent genes in p53AD vs p53 null. Table S4 shows p53half site analysis in PPAR target genes Table S5 shows control sequences used for p53 half site analysis Table S6 shows altered genes in p53AD-derived thymic lymphomas Table S7 shows primers used for genotyping of p53 mutant mice Table S8 shows primers used for qRT-PCR Table S9 shows antibodies used for flow cytometry Figure S1 shows extended data for generation of knock-in mouse models and features of p53TD mutant proteins and mice. Figure S2 shows homozygous mutant mice survival curves as well as tumor data. Figure S3 shows exome sequencing results for thymic lymphomas and hemangiosarcomas derived from p53 monomer and dimer mutant mice. Figure S4 shows characterization of stress responses of p53TD mutants. Figure S5 shows RNA-sequencing results for p53AD and p53RP, and Mdm2 genetic deletion rescue experiments. Figure S6 shows RNA-seq results of basal p53AD vs p53-null thymi, upstream regulator analysis, and ATAC-seq results for specific loci. Figure S7 shows extended data for p53 dimer mutant novel activities (co-IP, motif analysis, other dimer mutants activities, and tumor data) Figure S8 shows ferroptosis and scRNA-seq extended data Figure S9 shows diagram summarizing findings within this study.

Cancer-related alterations of the p53 tetramerization domain (TD) abrogate wild-type (WT) p53 function. They result in a protein that preferentially forms monomers or dimers, which are also normal p53 states under basal cellular conditions. However, their physiologic relevance is not well understood. We have established in vivo models for monomeric and dimeric p53, which model Li–Fraumeni syndrome patients with germline p53 TD alterations. p53 monomers are inactive forms of the protein. Unexpectedly, p53 dimers conferred some tumor suppression that is not mediated by canonical WT p53 activities. p53 dimers upregulate the PPAR pathway. These activities are associated with lower prevalence of thymic lymphomas and increased CD8+ T-cell differentiation. Lymphomas derived from dimeric p53 mice show cooperating alterations in the PPAR pathway, further implicating a role for these activities in tumor suppression. Our data reveal novel functions for p53 dimers and support the exploration of PPAR agonists as therapies.Significance:New mouse models with TP53R342P (monomer) or TP53A347D (dimer) mutations mimic Li–Fraumeni syndrome. Although p53 monomers lack function, p53 dimers conferred noncanonical tumor-suppressive activities. We describe novel activities for p53 dimers facilitated by PPARs and propose these are “basal” p53 activities.See related commentary by Stieg et al., p. 1046.See related article by Choe et al., p. 1250.This article is highlighted in the In This Issue feature, p. 1027

Table S1 shows predicted off target sites for p53TD mutant mice. Table S2 shows upregulated PPAR target genes in p53AD vs p53-null. Table S3 shows ATAC-seq and RNA-seq congruent genes in p53AD vs p53 null. Table S4 shows p53half site analysis in PPAR target genes Table S5 shows control sequences used for p53 half site analysis Table S6 shows altered genes in p53AD-derived thymic lymphomas Table S7 shows primers used for genotyping of p53 mutant mice Table S8 shows primers used for qRT-PCR Table S9 shows antibodies used for flow cytometry Figure S1 shows extended data for generation of knock-in mouse models and features of p53TD mutant proteins and mice. Figure S2 shows homozygous mutant mice survival curves as well as tumor data. Figure S3 shows exome sequencing results for thymic lymphomas and hemangiosarcomas derived from p53 monomer and dimer mutant mice. Figure S4 shows characterization of stress responses of p53TD mutants. Figure S5 shows RNA-sequencing results for p53AD and p53RP, and Mdm2 genetic deletion rescue experiments. Figure S6 shows RNA-seq results of basal p53AD vs p53-null thymi, upstream regulator analysis, and ATAC-seq results for specific loci. Figure S7 shows extended data for p53 dimer mutant novel activities (co-IP, motif analysis, other dimer mutants activities, and tumor data) Figure S8 shows ferroptosis and scRNA-seq extended data Figure S9 shows diagram summarizing findings within this study.

Cancer-related alterations of the p53 tetramerization domain (TD) abrogate wild-type (WT) p53 function. They result in a protein that preferentially forms monomers or dimers, which are also normal p53 states under basal cellular conditions. However, their physiologic relevance is not well understood. We have established in vivo models for monomeric and dimeric p53, which model Li–Fraumeni syndrome patients with germline p53 TD alterations. p53 monomers are inactive forms of the protein. Unexpectedly, p53 dimers conferred some tumor suppression that is not mediated by canonical WT p53 activities. p53 dimers upregulate the PPAR pathway. These activities are associated with lower prevalence of thymic lymphomas and increased CD8+ T-cell differentiation. Lymphomas derived from dimeric p53 mice show cooperating alterations in the PPAR pathway, further implicating a role for these activities in tumor suppression. Our data reveal novel functions for p53 dimers and support the exploration of PPAR agonists as therapies.Significance:New mouse models with TP53R342P (monomer) or TP53A347D (dimer) mutations mimic Li–Fraumeni syndrome. Although p53 monomers lack function, p53 dimers conferred noncanonical tumor-suppressive activities. We describe novel activities for p53 dimers facilitated by PPARs and propose these are “basal” p53 activities.See related commentary by Stieg et al., p. 1046.See related article by Choe et al., p. 1250.This article is highlighted in the In This Issue feature, p. 1027

Cancer-related alterations of the p53 tetramerization domain (TD) abrogate wild-type (WT) p53 function. They result in a protein that preferentially forms monomers or dimers, which are also normal p53 states under basal cellular conditions. However, their physiologic relevance is not well understood. We have established in vivo models for monomeric and dimeric p53, which model Li–Fraumeni syndrome patients with germline p53 TD alterations. p53 monomers are inactive forms of the protein. Unexpectedly, p53 dimers conferred some tumor suppression that is not mediated by canonical WT p53 activities. p53 dimers upregulate the PPAR pathway. These activities are associated with lower prevalence of thymic lymphomas and increased CD8+ T-cell differentiation. Lymphomas derived from dimeric p53 mice show cooperating alterations in the PPAR pathway, further implicating a role for these activities in tumor suppression. Our data reveal novel functions for p53 dimers and support the exploration of PPAR agonists as therapies.Significance:New mouse models with TP53R342P (monomer) or TP53A347D (dimer) mutations mimic Li–Fraumeni syndrome. Although p53 monomers lack function, p53 dimers conferred noncanonical tumor-suppressive activities. We describe novel activities for p53 dimers facilitated by PPARs and propose these are “basal” p53 activities.

Table S1 shows predicted off target sites for p53TD mutant mice. Table S2 shows upregulated PPAR target genes in p53AD vs p53-null. Table S3 shows ATAC-seq and RNA-seq congruent genes in p53AD vs p53 null. Table S4 shows p53half site analysis in PPAR target genes Table S5 shows control sequences used for p53 half site analysis Table S6 shows altered genes in p53AD-derived thymic lymphomas Table S7 shows primers used for genotyping of p53 mutant mice Table S8 shows primers used for qRT-PCR Table S9 shows antibodies used for flow cytometry Figure S1 shows extended data for generation of knock-in mouse models and features of p53TD mutant proteins and mice. Figure S2 shows homozygous mutant mice survival curves as well as tumor data. Figure S3 shows exome sequencing results for thymic lymphomas and hemangiosarcomas derived from p53 monomer and dimer mutant mice. Figure S4 shows characterization of stress responses of p53TD mutants. Figure S5 shows RNA-sequencing results for p53AD and p53RP, and Mdm2 genetic deletion rescue experiments. Figure S6 shows RNA-seq results of basal p53AD vs p53-null thymi, upstream regulator analysis, and ATAC-seq results for specific loci. Figure S7 shows extended data for p53 dimer mutant novel activities (co-IP, motif analysis, other dimer mutants activities, and tumor data) Figure S8 shows ferroptosis and scRNA-seq extended data Figure S9 shows diagram summarizing findings within this study.