Oncogene-induced senescence (OIS) is crucial for tumour suppression. Senescent cells implement a complex pro-inflammatory response termed the senescence-associated secretory phenotype (SASP). The ...SASP reinforces senescence, activates immune surveillance and paradoxically also has pro-tumorigenic properties. Here, we present evidence that the SASP can also induce paracrine senescence in normal cells both in culture and in human and mouse models of OIS in vivo. Coupling quantitative proteomics with small-molecule screens, we identified multiple SASP components mediating paracrine senescence, including TGF-β family ligands, VEGF, CCL2 and CCL20. Amongst them, TGF-β ligands play a major role by regulating p15(INK4b) and p21(CIP1). Expression of the SASP is controlled by inflammasome-mediated IL-1 signalling. The inflammasome and IL-1 signalling are activated in senescent cells and IL-1α expression can reproduce SASP activation, resulting in senescence. Our results demonstrate that the SASP can cause paracrine senescence and impact on tumour suppression and senescence in vivo.
Mammalian gene expression displays widespread circadian oscillations. Rhythmic transcription underlies the core clock mechanism, but it cannot explain numerous observations made at the level of ...protein rhythmicity. We have used ribosome profiling in mouse liver to measure the translation of mRNAs into protein around the clock and at high temporal and nucleotide resolution. We discovered, transcriptome-wide, extensive rhythms in ribosome occupancy and identified a core set of approximately 150 mRNAs subject to particularly robust daily changes in translation efficiency. Cycling proteins produced from nonoscillating transcripts revealed thus-far-unknown rhythmic regulation associated with specific pathways (notably in iron metabolism, through the rhythmic translation of transcripts containing iron responsive elements), and indicated feedback to the rhythmic transcriptome through novel rhythmic transcription factors. Moreover, estimates of relative levels of core clock protein biosynthesis that we deduced from the data explained known features of the circadian clock better than did mRNA expression alone. Finally, we identified uORF translation as a novel regulatory mechanism within the clock circuitry. Consistent with the occurrence of translated uORFs in several core clock transcripts, loss-of-function of Denr, a known regulator of reinitiation after uORF usage and of ribosome recycling, led to circadian period shortening in cells. In summary, our data offer a framework for understanding the dynamics of translational regulation, circadian gene expression, and metabolic control in a solid mammalian organ.
The daily gene expression oscillations that underlie mammalian circadian rhythms show striking differences between tissues and involve post-transcriptional regulation. Both aspects remain poorly ...understood. We have used ribosome profiling to explore the contribution of translation efficiency to temporal gene expression in kidney and contrasted our findings with liver data available from the same mice.
Rhythmic translation of constantly abundant messenger RNAs (mRNAs) affects largely non-overlapping transcript sets with distinct phase clustering in the two organs. Moreover, tissue differences in translation efficiency modulate the timing and amount of protein biosynthesis from rhythmic mRNAs, consistent with organ specificity in clock output gene repertoires and rhythmicity parameters. Our comprehensive datasets provided insights into translational control beyond temporal regulation. Between tissues, many transcripts show differences in translation efficiency, which are, however, of markedly smaller scale than mRNA abundance differences. Tissue-specific changes in translation efficiency are associated with specific transcript features and, intriguingly, globally counteracted and compensated transcript abundance variations, leading to higher similarity at the level of protein biosynthesis between both tissues.
We show that tissue specificity in rhythmic gene expression extends to the translatome and contributes to define the identities, the phases and the expression levels of rhythmic protein biosynthesis. Moreover, translational compensation of transcript abundance divergence leads to overall higher similarity at the level of protein production across organs. The unique resources provided through our study will serve to address fundamental questions of post-transcriptional control and differential gene expression in vivo.
Circular RNAs (circRNAs) are found across eukaryotes and can function in post-transcriptional gene regulation. Their biogenesis through a circle-forming backsplicing reaction is facilitated by ...reverse-complementary repetitive sequences promoting pre-mRNA folding. Orthologous genes from which circRNAs arise, overall contain more strongly conserved splice sites and exons than other genes, yet it remains unclear to what extent this conservation reflects purifying selection acting on the circRNAs themselves. Our analyses of circRNA repertoires from five species representing three mammalian lineages (marsupials, eutherians: rodents, primates) reveal that surprisingly few circRNAs arise from orthologous exonic loci across all species. Even the circRNAs from orthologous loci are associated with young, recently active and species-specific transposable elements, rather than with common, ancient transposon integration events. These observations suggest that many circRNAs emerged convergently during evolution – as a byproduct of splicing in orthologs prone to transposon insertion. Overall, our findings argue against widespread functional circRNA conservation.
Gene-expression programs define shared and species-specific phenotypes, but their evolution remains largely uncharacterized beyond the transcriptome layer
. Here we report an analysis of the ...co-evolution of translatomes and transcriptomes using ribosome-profiling and matched RNA-sequencing data for three organs (brain, liver and testis) in five mammals (human, macaque, mouse, opossum and platypus) and a bird (chicken). Our within-species analyses reveal that translational regulation is widespread in the different organs, in particular across the spermatogenic cell types of the testis. The between-species divergence in gene expression is around 20% lower at the translatome layer than at the transcriptome layer owing to extensive buffering between the expression layers, which especially preserved old, essential and housekeeping genes. Translational upregulation specifically counterbalanced global dosage reductions during the evolution of sex chromosomes and the effects of meiotic sex-chromosome inactivation during spermatogenesis. Despite the overall prevalence of buffering, some genes evolved faster at the translatome layer-potentially indicating adaptive changes in expression; testis tissue shows the highest fraction of such genes. Further analyses incorporating mass spectrometry proteomics data establish that the co-evolution of transcriptomes and translatomes is reflected at the proteome layer. Together, our work uncovers co-evolutionary patterns and associated selective forces across the expression layers, and provides a resource for understanding their interplay in mammalian organs.
The apical domain of epithelial cells is composed of distinct subdomains such as microvilli, primary cilia and a non‐protruding region. Using the cholesterol‐binding protein prominin‐1 as a specific ...marker of plasma membrane protrusions we have previously proposed the co‐existence of different cholesterol‐based lipid microdomains (lipid rafts) within the apical domain Röper, K., Corbeil, D. and Huttner, W.B. (2000), Retention of prominin in microvilli reveals distinct cholesterol‐based lipid microdomains in the apical plasma membrane. Nat. Cell Biol. 2, 582–592. To substantiate the hypothesis that the microvillar plasma membrane subdomains contain a distinct set of lipids compared to the planar portion we have investigated the distribution of prominin‐1 and two raft‐associated gangliosides GM1 and GM3 by fluorescence microscopy. GM1 was found to co‐localize with prominin‐1 on microvilli whereas GM3 was segregated from there suggesting its localization in the planar region. Regarding the primary cilium, overlapping fluorescent signals of GM1 or GM3 and prominin‐1 were observed. Thus, our data demonstrate that specific ganglioside‐enriched rafts are found in different apical subdomains and reveal that two plasma membrane protrusions with different structural bases (actin for the microvillus and tubulin for the cilium) are composed of distinct types of lipid.
Murine epidermal stem cells undergo alternate cycles of dormancy and activation, fuelling tissue renewal. However, only a subset of stem cells becomes active during each round of morphogenesis, ...indicating that stem cells coexist in heterogeneous responsive states. Using a circadian-clock reporter-mouse model, here we show that the dormant hair-follicle stem cell niche contains coexisting populations of cells at opposite phases of the clock, which are differentially predisposed to respond to homeostatic cues. The core clock protein Bmal1 modulates the expression of stem cell regulatory genes in an oscillatory manner, to create populations that are either predisposed, or less prone, to activation. Disrupting this clock equilibrium, through deletion of Bmal1 (also known as Arntl) or Per1/2, resulted in a progressive accumulation or depletion of dormant stem cells, respectively. Stem cell arrhythmia also led to premature epidermal ageing, and a reduction in the development of squamous tumours. Our results indicate that the circadian clock fine-tunes the temporal behaviour of epidermal stem cells, and that its perturbation affects homeostasis and the predisposition to tumorigenesis.
Human skin copes with harmful environmental factors that are circadian in nature, yet how circadian rhythms modulate the function of human epidermal stem cells is mostly unknown. Here we show that in ...human epidermal stem cells and their differentiated counterparts, core clock genes peak in a successive and phased manner, establishing distinct temporal intervals during the 24 hr day period. Each of these successive clock waves is associated with a peak in the expression of subsets of transcripts that temporally segregate the predisposition of epidermal stem cells to respond to cues that regulate their proliferation or differentiation, such as TGFβ and calcium. Accordingly, circadian arrhythmia profoundly affects stem cell function in culture and in vivo. We hypothesize that this intricate mechanism ensures homeostasis by providing epidermal stem cells with environmentally relevant temporal functional cues during the course of the day and that its perturbation may contribute to aging and carcinogenesis.
Display omitted
•Core clock genes peak in a successive and phased manner in human epidermal SCs•Peaks establish distinct functional intervals during the 24 hr day period•Predisposition of hEpSCs to proliferate or differentiate segregates with the peaks•Circadian arrhythmia profoundly affects hEpSC function in culture and in vivo
The circadian clock regulates human epidermal stem cell function, connecting stem cell activity to relevant daily changes in the external environment.
Prominins (proms) are transmembrane glycoproteins conserved throughout the animal kingdom. They are associated with plasma membrane protrusions, such as primary cilia, as well as extracellular ...vesicles derived thereof. Primary cilia host numerous signaling pathways affected in diseases known as ciliopathies. Human PROM1 (CD133) is detected in both somatic and cancer stem cells and is also expressed in terminally differentiated epithelial and photoreceptor cells. Genetic mutations in the PROM1 gene result in retinal degeneration by impairing the proper formation of the outer segment of photoreceptors, a modified cilium. Here, we investigated the impact of proms on two distinct examples of ciliogenesis. First, we demonstrate that the overexpression of a dominant-negative mutant variant of human PROM1 (i.e. mutation Y819F/Y828F) significantly decreases ciliary length in Madin–Darby canine kidney cells. These results contrast strongly to the previously observed enhancing effect of WT PROM1 on ciliary length. Mechanistically, the mutation impeded the interaction of PROM1 with ADP-ribosylation factor–like protein 13B, a key regulator of ciliary length. Second, we observed that in vivo knockdown of prom3 in zebrafish alters the number and length of monocilia in the Kupffer's vesicle, resulting in molecular and anatomical defects in the left-right asymmetry. These distinct loss-of-function approaches in two biological systems reveal that prom proteins are critical for the integrity and function of cilia. Our data provide new insights into ciliogenesis and might be of particular interest for investigations of the etiologies of ciliopathies.
Human epidermal stem cells transit from a slow cycling to an actively proliferating state to contribute to homeostasis. Both stem cell states differ in their cell cycle profiles but must remain ...guarded from differentiation and senescence. Here we show that Cbx4, a Polycomb Repressive Complex 1 (PRC1)-associated protein, maintains human epidermal stem cells as slow-cycling and undifferentiated, while protecting them from senescence. Interestingly, abrogating the polycomb activity of Cbx4 impairs its antisenescent function without affecting stem cell differentiation, indicating that differentiation and senescence are independent processes in human epidermis. Conversely, Cbx4 inhibits stem cell activation and differentiation through its SUMO ligase activity. Global transcriptome and chromatin occupancy analyses indicate that Cbx4 regulates modulators of epidermal homeostasis and represses factors such as Ezh2, Dnmt1, and Bmi1 to prevent the active stem cell state. Our results suggest that distinct Polycomb complexes balance epidermal stem cell dormancy and activation, while continually preventing senescence and differentiation.
Display omitted
► Cbx4 prevents human epSC proliferation, senescence, and differentiation ► Cbx4 regulates senescence solely via PRC1 and differentiation via its SUMO activity ► Cbx4 is the only PRC1-associated Cbx protein to promote hepSC quiescence ► Differentiation and senescence are two independent processes in human epidermis
PDF
