Dataset Information

TP53 Haploinsufficiency Rescues Emergency Granulopoiesis in FANCC-/- Mice.

ABSTRACT: Emergency (stress) granulopoiesis is an episodic process for the production of granulocytes in response to infectious challenge. We previously determined that Fanconi C, a component of the Fanconi DNA-repair pathway, is necessary for successful emergency granulopoiesis. Fanconi anemia results from mutation of any gene in this pathway and is characterized by bone marrow failure (BMF) in childhood and clonal progression in adolescence. Although murine Fanconi anemia models exhibit relatively normal steady-state hematopoiesis, FANCC-/- mice are unable to mount an emergency granulopoiesis response. Instead, these mice develop BMF and die during repeated unsuccessful emergency granulopoiesis attempts. In FANCC-/- mice, BMF is associated with extensive apoptosis of hematopoietic stem and progenitor cells through an undefined mechanism. In this study, we find that TP53 haploinsufficiency completely rescues emergency granulopoiesis in FANCC-/- mice and protects them from BMF during repeated emergency granulopoiesis episodes. Instead, such recurrent challenges accelerated clonal progression in FANCC-/-TP53+/- mice. In FANCC-/- mice, BMF during multiple emergency granulopoiesis attempts was associated with increased ataxia telangiectasia and Rad3-related protein (Atr) and p53 activation with each attempt. In contrast, we found progressive attenuation of expression and activity of Atr, and consequent p53 activation and apoptosis, in the bone marrow of FANCC-/-TP53+/- mice during this process. Therefore, activation of Atr-with consequent Fanconi-mediated DNA repair or p53-dependent apoptosis-is an essential component of emergency granulopoiesis and it protects the bone marrow from genotoxic stress during this process.

SUBMITTER: Hu L

PROVIDER: S-EPMC5834788 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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TP53 Haploinsufficiency Rescues Emergency Granulopoiesis in FANCC-/- Mice.

Hu Liping L Huang Weiqi W Bei Ling L Broglie Larisa L Eklund Elizabeth A EA

Journal of immunology (Baltimore, Md. : 1950) 20180202 6

Emergency (stress) granulopoiesis is an episodic process for the production of granulocytes in response to infectious challenge. We previously determined that Fanconi C, a component of the Fanconi DNA-repair pathway, is necessary for successful emergency granulopoiesis. Fanconi anemia results from mutation of any gene in this pathway and is characterized by bone marrow failure (BMF) in childhood and clonal progression in adolescence. Although murine Fanconi anemia models exhibit relatively norma ...[more]

PMID: 29427417

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Project description:BACKGROUND: Previously we have suggested that cancer cells develop a mechanism(s) which allows for either: silencing of the wild-type TP53 transcription, degradation of the wild-type TP53 mRNA, or selective overproduction of the mutated TP53 mRNA, which is the subject of this article. Sequencing of TP53 on the respective cDNA and DNA templates from tumor samples were found to give discordant results. DNA analysis showed a pattern of heterozygous mutations, whereas the analysis of cDNA demonstrated the mutated template only. We hypothesized that different TP53 gene expression levels of each allele may be caused by the polymorphism within intron 3 (PIN3). The aim of this study was to test if one of the polymorphic variants of PIN3 (A1 or A2) in the heterozygotes is associated with a higher TP53 expression, and therefore, responsible for the haploinsufficiency phenomenon. METHODS: 250 tumor samples were tested. To analyze the involvement of PIN3 polymorphic variant (A1 or A2) on TP53 mRNA expression regulation, bacterial subcloning combined with sequencing analyses, dual luciferase reporter assays and bioinformatic analysis were performed. RESULTS: Haplotype analysis showed the predominance of the mutated template during the cDNA sequencing in all samples showing a heterozygous TP53 mutation and PIN3 heterozygosity. Out of 30 samples (from the total of 250 tested samples) which carried TP53 mutations and had a bias in allelic expression 6 were heterozygous for the A1/A2 polymorphism, and all 6 (p = 0.04) samples carried the mutation within the PIN3 longer allele (A2). Reporter assays revealed higher luciferase activity in cells transfected with the plasmid containing A2 construct than A1 and control. A2/A1 ratio ranged from 1.16 for AD293 cell line (p = 0.019) to 1.59 for SW962 cell line (p = 0.0019). Moreover, bioinformatic analyses showed that PIN3 duplication stabilized secondary DNA structures - G-quadruplexes. CONCLUSION: TP53 alleles are not equivalent for their impact on the regulation of expression of TP53 mRNA. Therefore, in PIN3-heterozygous cases a single TP53 mutation of the longer allele might sufficiently destabilize its function. Secondary DNA structures such as quadruplexes can also play a role in PIN3-dependent TP53 haploinsufficiency.

Project description:The current model of hematopoiesis only resolves the thymus around the production and establishment of the peripheral T cell pool. However, the role and in situ development of other immune subsets has been overlooked. We aim to demonstrate that during TL1A and IL-18 induced inflammation, the thymus is capable of producing other cell types such as neutrophils, monocytes, and macrophages and that these potentially have other functions aside from supporting T cell development as scavengers. Here we have proven that ex vivo and in vivo treatment with TL1A and IL-18 results in acute thymic atrophy by a massive loss of DP T cells and shrinking of the thymic lobe. By electron microscopy, flow cytometry and single cell we demonstrate that neutrophils are able to maturate inside the thymus lobe in a NOTCH-independent manner. We used fate-mapping tools to elucidate the origin of thymic neutrophils since we observed a great expansion in our culture that is isolated from the influx of BM progenitors. The Rag1-Cre Rosa26-YFP fate mapping model revealed that neutrophils share a common progenitor with T cells, while monocytes/macrophages do not. Furthermore, we found that most of the thymic GMPs (defined as Lin-Sca-1-c-Kit+CD16/32+CD34+) show history of Rag1 expression in comparison with bone marrow GMPs. Additionally, by using Ms4a3-Cre Rosa26Tdtomato fate mapping model we observed that there was not difference in Tdt labeling between the thymus and bone marrow, suggesting that thymic neutrophils still undergo conventional neutrophils pathway arising from GMPs. Moreover, we show that thymic-derived neutrophils are functional and are capable of forming extracellular neutrophils traps (NETs) similarly to benchmark peritoneal neutrophils. We found that the expansion of thymic neutrophils is GM-CSF dependent by using Csf2rb KO mice. Additionally, we identified DR3+ and IL-18Rα+ expressing subsets of ILCs and gdT cells as the cellular source GM-CSF. Lastly, in vivo treatment with TL1A+IL-18 lead to emergency granulopoiesis and an increase of neutrophils in all the organs investigated, including the thymus.

Dataset Information

TP53 Haploinsufficiency Rescues Emergency Granulopoiesis in FANCC-/- Mice.

Publications

<i>TP53</i> Haploinsufficiency Rescues Emergency Granulopoiesis in <i>FANCC</i><sup>-/-</sup> Mice.

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