Project description:The immune system consists of a network of specialized cells and processes that protect the organism from diseases and regulates tissue repair. Natural killer (NK) cells and the αβ and γδ T cells comprise the cytotoxic arm of the immune system and have evolved to detect and eradicate cells infected with pathogens and affected by malignancies, in order to prevent their spread and subsequent tissue damage. The naked mole-rat (NMR; Heterocephalus glaber) exhibits an unusually long lifespan relative to its body size, remarkable cancer resistance, and a surprising absence of NK cells, likely lost during its evolution. So far, little is known about NMR T cells in terms of the subsets they comprise, the evolution of the genes that regulate their function, the diversity of their clonotypes, and the organ in which they mature – the thymus. Using single-cell RNA sequencing (scRNA-seq), we find that NMRs have a large population of circulating γδT cells, comprising two subsets, one with an activated cytotoxic profile. Using comparative genomics, we find that the NMR genome maintains a large diversity of γ and δ variable T-cell receptor (TCR) regions, relative to that in other mammalian genomes. We devise a novel TCR scRNA-seq approach, which reveals that the NMR cytotoxic γδT-cell subset harbors a dominant clonotype, and that, in contrast to mouse, the clonotypic diversity of NMR CD8 αβT cells is significantly lower than that of its CD4 αβT cells. The latter suggests that NMRs have evolved under a relaxed intracellular pathogenic selective pressure, consistent with the loss of their NK cells. We corroborate this by showing that the genomic diversity of NMR MHC-I genes, relative to that of MHC-II genes, is considerably smaller than in other mammalian genomes. In line with these findings, we further show that NMR thymi in early life are considerably smaller compared to mouse thymi, yet by middle age thymi of both species show equivalent signs of involution. Our study thus sheds significant light on NMR T cells and moves us one step closer towards understanding the contributions of the immune system to the remarkable longevity and cancer resistance of this species.

Project description:The naked mole-rat (NMR; Heterocephalus glaber) has recently gained considerable attention in the scientific community for its unique potential to unveil novel insights in the fields of medicine, biochemistry, and evolution. NMRs exhibit unique adaptations that include protracted fertility, cancer resistance, eusociality, and anoxia. This suite of adaptations is not found in other rodent species, suggesting that interrogating conserved and accelerated regions in the NMR genome will find regions of the NMR genome fundamental to their unique adaptations. However, the current NMR genome assembly has limits that make studying structural variations, heterozygosity, and non-coding adaptations challenging. We present a complete diploid naked-mole rat genome assembly by integrating long-read and 10X-linked read genome sequencing of a male NMR and its parents, and Hi-C sequencing in the NMR hypothalamus (N=2). Reads were identified as maternal, paternal or ambiguous (TrioCanu). We then polished genomes with Flye, Racon and Medaka. Assemblies were then scaffolded using the following tools in order: Scaff10X, Salsa2, 3d-DNA, Minimap2-alignment between assemblies, and the Juicebox Assembly Tools. We then subjected the assemblies to another round of polishing, including short-read polishing with Freebayes. We assembled the NMR mitochondrial genome with mitoVGP. Y chromosome contigs were identified by aligning male and female 10X linked reads to the paternal genome and finding male-biased contigs not present in the maternal genome. Contigs were assembled with publicly available male NMR Fibroblast Hi-C-seq data (SRR820318). Both assemblies have their sex chromosome haplotypes merged so that both assemblies have a high-quality X and Y chromosome. Finally, assemblies were evaluated with Quast, BUSCO, and Merqury, which all reported the base-pair quality and contiguity of both assemblies as high-quality. The assembly will next be annotated by Ensembl using public RNA-seq data from multiple tissues (SRP061363). Together, this assembly will provide a high-quality resource to the NMR and comparative genomics communities.

Project description:The naked mole-rat, Heterocephalus glaber (NMR), the longest-lived rodent with a maximum possible lifespan exceeding 33 years, is emerging as an important non-model organism for the study of longevity and healthspan. As such it is of significance and interest in the study of biomarkers for ageing in mammals. Recent breakthroughs in this field have indicated that ‘epigenetic clocks’ based on the temporal accumulation of DNA methylation at specific genomic sites can enable accurate age estimates for tissues across the lifespan of an individual. Here, we validate the hypothesis of an epigenetic clock in NMRs, and create a method for predicting the age of naked mole-rats based on changes in methylation of targeted CpG sites in regions known as ageing-associated differentially methylated positions (aDMPs). In the discovery phase, we performed a targeted analysis of 51 different CpGs in 24 different NMR livers spanning an age range from 39 weeks to 1,144 weeks. Of these 51 different sites, 23 were found to be significantly associated with age (p < 0.05). We then built a predictor of age using the 23 sites that showed an association with age. To test the accuracy of this model, we predicted age in an additional test set of 19 different livers spanning an age range 43 to 1,196 weeks. Our model was able to successfully predict age in the test with a root mean squared error (RMSE) of 166.11 weeks. We also profiled a 20 skin samples with the same age range and found a striking correlation between the predicted age of these samples versus their actual age (R=0.93). However, this correlation when compared to the liver samples showed a lower predicted age than actual age, suggesting that skin tissue ages slower than the liver in NMRs. Finally, we have produced freely available software tool that will take in raw sequencing data and produce an age prediction for new NMR samples. Our model will enable the prediction of age in wild caught naked mole-rats and captive animals of unknown age, and will be invaluable for further mechanistic studies of mammalian ageing.

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Age-associated changes in the skin transcriptome in Naked mole-rats

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