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Head and Neck Squamous Cell Carcinoma (HNSCC) remains among the most aggressive human cancers. Tumor progression and aggressiveness in SCC are largely driven by Tumor Propagating Cells (TPCs). Aerobic glycolysis, also known as the Warburg Effect, represents a characteristic of many cancers, yet whether this adaptation is functionally important in SCC, and at which stage, remains poorly understood. Here, we show that the NAD+-dependent histone deacetylase Sirtuin 6 (SIRT6) is a robust tumor suppressor in SCC, acting as a modulator of glycolysis in these tumors. Remarkably, rather than a late adaptation, we find enhanced glycolysis specifically in TPCs. More importantly, using single cell RNA sequencing of TPCs, we identify a subset of TPCs with higher glycolysis and enhanced pentose phosphate pathway and glutathione metabolism, characteristics that are strongly associated with a better antioxidant response. Altogether, our studies uncover enhanced glycolysis as a main driver in SCC, and, more importantly, identify a subset of TPCs as the cell-of-origin for the Warburg effect, defining metabolism as a key feature of intra-tumor heterogeneity.

© The Author(s) 2020., Regenerative proliferation capacity and poor differentiation are histological features usually linked to poor prognosis in head and neck squamous cell carcinoma (hnSCC). However, the pathways that regulate them remain ill-characterized. Here, we show that those traits can be triggered by the RHO GTPase activator VAV2 in keratinocytes present in the skin and oral mucosa. VAV2 is also required to maintain those traits in hnSCC patient-derived cells. This function, which is both catalysis- and RHO GTPase-dependent, is mediated by c-Myc- and YAP/TAZ-dependent transcriptomal programs associated with regenerative proliferation and cell undifferentiation, respectively. High levels of VAV2 transcripts and VAV2-regulated gene signatures are both associated with poor hnSCC patient prognosis. These results unveil a druggable pathway linked to the malignancy of specific SCC subtypes., We thank M. Blázquez and CIC facilities’ personnel for lab work and core unit technical assistance, respectively. X.R.B. is supported by grants from Worldwide Cancer Research (14-1248), the Castilla-León Government (CSI049U16, CLC-2017-01), the Spanish Ministry of Science and Innovation (MSI) (SAF2015-64556-R, RTI2018-096481-B-I00), the Ramón Areces Foundation, and the Spanish Association against Cancer (GC16173472GARC). X.R.B.’s institution is supported by the Programa de Apoyo a Planes Estratégicos de Investigación de Estructuras de Investigación de Excelencia of the Ministry of Education of the Castilla-León Government (CLC-2017-01). J.M.P. is supported by MSI grants (SAF2015-66015-R, PIE15/00076). S.A.B. was supported by the European Research Council, the Spanish MSIU, and the IRB-Barcelona. S.R.-F. and L.F. L.-M. contracts have been supported by funding from the MSI (BES-2013-063573) and the Spanish Ministry of Education, Culture, and Sports (L.F.L.-M., FPU13/02923), respectively. Both Spanish and Castilla-León government-associated funding is partially supported by the European Regional Development Fund.

Tissue regeneration requires coordination between resident stem cells and local niche cells1,2. Here we identify that senescent cells are integral components of the skeletal muscle regenerative niche that repress regeneration at all stages of life. The technical limitation of senescent-cell scarcity3 was overcome by combining single-cell transcriptomics and a senescent-cell enrichment sorting protocol. We identified and isolated different senescent cell types from damaged muscles of young and old mice. Deeper transcriptome, chromatin and pathway analyses revealed conservation of cell identity traits as well as two universal senescence hallmarks (inflammation and fibrosis) across cell type, regeneration time and ageing. Senescent cells create an aged-like inflamed niche that mirrors inflammation associated with ageing (inflammageing4) and arrests stem cell proliferation and regeneration. Reducing the burden of senescent cells, or reducing their inflammatory secretome through CD36 neutralization, accelerates regeneration in young and old mice. By contrast, transplantation of senescent cells delays regeneration. Our results provide a technique for isolating in vivo senescent cells, define a senescence blueprint for muscle, and uncover unproductive functional interactions between senescent cells and stem cells in regenerative niches that can be overcome. As senescent cells also accumulate in human muscles, our findings open potential paths for improving muscle repair throughout life. We thank M. Jardí, A. Navarro, J. M. Ballestero, K. Slobodnyuk, M. González, J. López and M. Raya for their technical contributions; A. Harada and K. Tanaka for assistance in ATAC-seq; all of the members of the P.M.-C. laboratory for discussions; J. Campisi for p16-3MR mice; J. A. Fernández-Blanco (PRBB Animal Facility); O. Fornas (UPF/CRG FACS Facility); E. Rebollo (IBMB Molecular Imaging Platform); V. A. Raker for manuscript editing; and the members of the Myoage network (A. Maier) for human material. We acknowledge funding from MINECO-Spain (RTI2018-096068, to P.M.-C. and E.P.); ERC-2016-AdG-741966, LaCaixa-HEALTH-HR17-00040, MDA, UPGRADE-H2020-825825, AFM, DPP-Spain, Fundació La MaratóTV3-80/19-202021 and MWRF to P.M.-C.; Fundació La MaratóTV3-137/38-202033 to A.L.S.; María-de-Maeztu Program for Units of Excellence to UPF (MDM-2014-0370) and Severo-Ochoa Program for Centers of Excellence to CNIC (SEV-2015-0505). This work was also supported by JST-CREST JPMJCR16G1 and MEXT/JSPS JP20H00456/18H05527 to Y.O.; the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16030502) to M.A.E.; V.M. and A.C. were supported by FPI and Maria-de-Maeztu predoctoral fellowships, respectively, and V.S. by a Marie Skłodowska-Curie individual fellowship. Parts of the figures were drawn using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licences/by/3.0/).

Summary Molecular daily rhythms can be captured by precisely timed tissue harvests from groups of animals. This protocol will allow the investigator to identify transcriptional rhythms in the mouse liver while also providing a template for similar analyses in other whole metabolic organs. We describe steps for mouse entrainment, liver dissection, and rhythmicity analysis from total RNA sequencing data. The resulting rhythmic transcriptome will provide the user with a starting point for defining specific biological processes that undergo daily rhythms. For complete details on the use and execution of this protocol, please refer to Koronowski et al. (2019). A similar protocol for interfollicular epidermal cells is demonstrated in Welz et al. (2019)., Graphical abstract, Highlights • Investigate a gene or pathway in the fourth dimension—time • Use precisely timed tissue harvest to detect daily transcriptional rhythms • Full description of mouse entrainment, liver dissection, and rhythmicity analysis • A reference protocol for investigating other whole metabolic organs, Molecular daily rhythms can be captured by precisely timed tissue harvests from groups of animals. This protocol will allow the investigator to identify transcriptional rhythms in the mouse liver while also providing a template for similar analyses in other whole metabolic organs. We describe steps for mouse entrainment, liver dissection, and rhythmicity analysis from total RNA sequencing data. The resulting rhythmic transcriptome will provide the user with a starting point for defining specific biological processes that undergo daily rhythms.

Fatty acid uptake and altered metabolism constitute hallmarks of metastasis1,2, yet evidence of the underlying biology, as well as whether all dietary fatty acids are prometastatic, is lacking. Here we show that dietary palmitic acid (PA), but not oleic acid or linoleic acid, promotes metastasis in oral carcinomas and melanoma in mice. Tumours from mice that were fed a short-term palm-oil-rich diet (PA), or tumour cells that were briefly exposed to PA in vitro, remained highly metastatic even after being serially transplanted (without further exposure to high levels of PA). This PA-induced prometastatic memory requires the fatty acid transporter CD36 and is associated with the stable deposition of histone H3 lysine 4 trimethylation by the methyltransferase Set1A (as part of the COMPASS complex (Set1A/COMPASS)). Bulk, single-cell and positional RNA-sequencing analyses indicate that genes with this prometastatic memory predominantly relate to a neural signature that stimulates intratumoural Schwann cells and innervation, two parameters that are strongly correlated with metastasis but are aetiologically poorly understood3,4. Mechanistically, tumour-associated Schwann cells secrete a specialized proregenerative extracellular matrix, the ablation of which inhibits metastasis initiation. Both the PA-induced memory of this proneural signature and its long-term boost in metastasis require the transcription factor EGR2 and the glial-cell-stimulating peptide galanin. In summary, we provide evidence that a dietary metabolite induces stable transcriptional and chromatin changes that lead to a long-term stimulation of metastasis, and that this is related to a proregenerative state of tumour-activated Schwann cells. Palmitic acid induces stable transcriptional and chromatin changes that lead to long-term stimulation of metastasis in orthotopic models of cancer through the secretion by tumour-associated Schwann cells of a specialized proregenerative extracellular matrix, the ablation of which inhibits metastasis initiation.

The role of heterochromatin in cell fate specification during development is unclear. We demonstrate that loss of the lysine 9 of histone H3 (H3K9) methyltransferase G9a in the mammary epithelium results in de novo chromatin opening, aberrant formation of the mammary ductal tree, impaired stem cell potential, disrupted intraductal polarity, and loss of tissue function. G9a loss derepresses long terminal repeat (LTR) retroviral sequences (predominantly the ERVK family). Transcriptionally activated endogenous retroviruses generate double-stranded DNA (dsDNA) that triggers an antiviral innate immune response, and knockdown of the cytosolic dsDNA sensor Aim2 in G9a knockout (G9acKO) mammary epithelium rescues mammary ductal invasion. Mammary stem cell transplantation into immunocompromised or G9acKO-conditioned hosts shows partial dependence of the G9acKO mammary morphological defects on the inflammatory milieu of the host mammary fat pad. Thus, altering the chromatin accessibility of retroviral elements disrupts mammary gland development and stem cell activity through both cell-autonomous and non-autonomous mechanisms.

How the proto-oncogene c-Myc balances the processes of stem-cell self-renewal, proliferation and differentiation in adult tissues is largely unknown. We explored c-Myc's transcriptional roles at the epidermal differentiation complex, a locus essential for skin maturation. Binding of c-Myc can simultaneously recruit (Klf4, Ovol-1) and displace (Cebpa, Mxi1 and Sin3a) specific sets of differentiation-specific transcriptional regulators to epidermal differentiation complex genes. We found that Sin3a causes deacetylation of c-Myc protein to directly repress c-Myc activity. In the absence of Sin3a, genomic recruitment of c-Myc to the epidermal differentiation complex is enhanced, and re-activation of c-Myc-target genes drives aberrant epidermal proliferation and differentiation. Simultaneous deletion of c-Myc and Sin3a reverts the skin phenotype to normal. Our results identify how the balance of two transcriptional key regulators can maintain tissue homeostasis through a negative feedback loop.

The mammalian circadian clockwork has evolved as a timing system that allows the daily environmental changes to be anticipated so that behavior and tissue physiology can be adjusted accordingly. The circadian clock synchronizes the function of all cells within tissues in order to temporally separate preclusive and potentially harmful physiologic processes and to establish a coherent temporal organismal physiology. Thus, the proper functioning of the circadian clockwork is essential for maintaining cellular and tissue homeostasis. Importantly, aging reduces the robustness of the circadian clock, resulting in disturbed sleep–wake cycles, a lowered capacity to synchronize circadian rhythms in peripheral tissues, and reprogramming of the circadian clock output at the molecular function levels. These circadian clock–dependent behavioral and molecular changes in turn further accelerate the process of aging. Here we review the current knowledge about how aging affects the circadian clock, how the functional decline of the circadian clock affects aging, and how the circadian clock machinery and the molecular processes that underlie aging are intertwined.

Summary Circadian rhythms control organismal physiology throughout the day. At the cellular level, clock regulation is established by a self-sustained Bmal1-dependent transcriptional oscillator network. However, it is still unclear how different tissues achieve a synchronized rhythmic physiology. That is, do they respond independently to environmental signals, or require interactions with each other to do so? We show that unexpectedly, light synchronizes the Bmal1-dependent circadian machinery in single tissues in the absence of Bmal1 in all other tissues. Strikingly, light-driven tissue autonomous clocks occur without rhythmic feeding behavior and are lost in constant darkness. Importantly, tissue-autonomous Bmal1 partially sustains homeostasis in otherwise arrhythmic and prematurely aging animals. Our results therefore support a two-branched model for the daily synchronization of tissues: an autonomous response branch, whereby light entrains circadian clocks without any commitment of other Bmal1-dependent clocks, and a memory branch using other Bmal1-dependent clocks to “remember” time in the absence of external cues.

Metastasis remains the leading cause of cancer-related deaths worldwide, and our inability to identify the tumour cells that colonize distant sites hampers the development of effective anti-metastatic therapies. However, with recent research advances we are beginning to distinguish metastasis-initiating cells from their non-metastatic counterparts. Importantly, advances in genome sequencing indicate that the acquisition of metastatic competency does not involve the progressive accumulation of driver mutations; moreover, in the early stages of tumorigenesis, cancer cells harbour combinations of driver mutations that endow them with metastatic competency. Novel findings highlight that cells can disseminate to distant sites early during primary tumour growth, remaining dormant and untreatable for long periods before metastasizing. Thus, metastatic cells must require local and systemic influences to generate metastases. This hypothesis suggests that factors derived from our lifestyle, such as our diet, exert a strong influence on tumour progression, and that such factors could be modulated if understood. Here, we summarize the recent findings on how specific metabolic cues modulate the behaviour of metastatic cells and how they influence the genome and epigenome of metastatic cells. We also discuss how crosstalk between metabolism and the epigenome can be harnessed to develop new anti-metastatic therapies., Summary: In this Review the authors summarize recent findings on how specific metabolic cues modulate and influence the behaviour, genome and epigenome of metastatic cancer cells. They also discuss how the crosstalk between metabolism and the epigenome of metastatic cells can be harnessed to develop new anti-metastatic therapies.

Mutations in, and the altered expression of, epigenetic modifiers are pervasive in human tumours, making epigenetic factors attractive antitumour targets. The open-versus-closed chromatin state within the cells-of-origin of cancer correlates with the uneven distribution of mutations. However, the long-term effect of targeting epigenetic modifiers on mutability in patients with cancer is unclear. Here, we increased chromatin accessibility by deleting the histone H3 lysine 9 (H3K9) methyltransferase G9a in murine epidermis and show that this does not alter the single nucleotide variant burden or global genomic distribution in chemical mutagen-induced squamous tumours. G9a-depleted tumours develop after a prolonged latency compared with their wild-type counterparts, but are more aggressive and have an expanded cancer progenitor pool, pronounced genomic instability and frequent loss-of-function p53 mutations. Thus, we call for caution when assessing long-term therapeutic benefits of chromatin modifier inhibitors, which may promote more aggressive disease.

The chronic skin inflammation psoriasis is crucially dependent on the IL-23/IL-17 cytokine axis. Although IL-23 is expressed by psoriatic keratinocytes and immune cells, only the immune cell-derived IL-23 is believed to be disease relevant. Here we use a genetic mouse model to show that keratinocyte-produced IL-23 is sufficient to cause a chronic skin inflammation with an IL-17 profile. Furthermore, we reveal a cell-autonomous nuclear function for the actin polymerizing molecule N-WASP, which controls IL-23 expression in keratinocytes by regulating the degradation of the histone methyltransferases G9a and GLP, and H3K9 dimethylation of the IL-23 promoter. This mechanism mediates the induction of IL-23 by TNF, a known inducer of IL-23 in psoriasis. Finally, in keratinocytes of psoriatic lesions a decrease in H3K9 dimethylation correlates with increased IL-23 expression, suggesting relevance for disease. Taken together, our data describe a molecular pathway where epigenetic regulation of keratinocytes can contribute to chronic skin inflammation., Although IL-23 is expressed by psoriatic keratinocytes as well as immune cells, only the immune cell derived IL-23 is thought to be important for the development of psoriasis. Here the authors provide evidence that keratinocyte-produced IL-23 is sufficient to cause a chronic skin inflammation.

The circadian timekeeping mechanism adapts physiology to the 24-hour light/dark cycle. However, how the outputs of the circadian clock in different peripheral tissues communicate and synchronize each other is still not fully understood. The circadian clock has been implicated in the regulation of numerous processes, including metabolism, the cell cycle, cell differentiation, immune responses, redox homeostasis, and tissue repair. Accordingly, perturbation of the machinery that generates circadian rhythms is associated with metabolic disorders, premature ageing, and various diseases including cancer. Importantly, it is now possible to target circadian rhythms through systemic or local delivery of time cues or compounds. Here, we summarize recent findings in peripheral tissues that link the circadian clock machinery to tissue-specific functions and diseases.

For many patients with breast cancer, symptomatic bone metastases appear after years of latency. How micrometastatic lesions remain dormant and undetectable before initiating colonization is unclear. Here, we describe a mechanism involved in bone metastatic latency of oestrogen receptor-positive (ER

The DNA methyltransferase Dnmt3a suppresses tumorigenesis in models of leukemia and lung cancer. Conversely, deregulation of Dnmt3b is thought to generally promote tumorigenesis. However, the role of Dnmt3a and Dnmt3b in many types of cancer remains undefined. Here, we show that Dnmt3a and Dnmt3b are dispensable for homeostasis of the murine epidermis. However, loss of Dnmt3a-but not Dnmt3b-increases the number of carcinogen-induced squamous tumors, without affecting tumor progression. Only upon combined deletion of Dnmt3a and Dnmt3b, squamous carcinomas become more aggressive and metastatic. Mechanistically, Dnmt3a promotes the expression of epidermal differentiation genes by interacting with their enhancers and inhibits the expression of lipid metabolism genes, including PPAR-γ, by directly methylating their promoters. Importantly, inhibition of PPAR-γ partially prevents the increase in tumorigenesis upon deletion of Dnmt3a. Altogether, we demonstrate that Dnmt3a and Dnmt3b protect the epidermis from tumorigenesis and that squamous carcinomas are sensitive to inhibition of PPAR-γ. DOI: http://dx.doi.org/10.7554/eLife.21697.001, eLife digest Most of the cells in our body contain the same DNA. However, our bodies are made of many different types of cell, such as nerve cells or skin cells, which perform very different jobs. In each cell type only certain sets of genes encoded by the DNA are active. Proteins known as epigenetic regulators are responsible for producing the different patterns of gene activity. If epigenetic regulators are switched on or off at the wrong time, they can contribute to ageing and diseases such as cancer. Enzymes known as DNA methyltransferases are one group of epigenetic regulators. DNA methyltransferases control the activity of genes by adding small chemical groups known as methyl groups to the DNA. Two of these enzymes – known as Dnmt3a and Dnmt3b – are important during development to help cells mature and specialize into different types. Mice that lack both of these enzymes either die as embryos or just after birth. Furthermore, these enzymes are mutated or less active in some skin cancers and various other human cancers. Here, Rinaldi et al. investigated the role these enzymes play in adult mice. The experiments show that under ordinary laboratory conditions, mutant mice that lacked Dnmt3a and Dnmt3b were as healthy as normal mice. However, when the mice were exposed to chemicals that promote tumor growth, which mimics skin exposure to UV light, the mutant mice developed many more skin tumors than the normal mice. Furthermore, the tumors in the mutant mice were more likely to form secondary tumors in the lung. Rinaldi et al. found that Dnmt3a reduced the production of a protein called PPAR-γ, which helps to break down some types of fat molecules. Treating the mutant mice with a drug that inhibits PPAR-γ activity slowed the growth of the tumors. Overall, these experiments show a new way in which DNA methyltransferases act in adult animals. Future research will investigate whether drugs that inhibit the breakdown of fats could help to treat cancers in which the Dnmt3a and Dnmt3b proteins are mutated or less active. DOI: http://dx.doi.org/10.7554/eLife.21697.002

Ovarian cancer (OvCa) is characterized by widespread and rapid metastasis in the peritoneal cavity. Visceral adipocytes promote this process by providing fatty acids (FAs) for tumour growth. However, the exact mechanism of FA transfer from adipocytes to cancer cells remains unknown. This study shows that OvCa cells co-cultured with primary human omental adipocytes express high levels of the FA receptor, CD36, in the plasma membrane, thereby facilitating exogenous FA uptake. Depriving OvCa cells of adipocyte-derived FAs using CD36 inhibitors and short hairpin RNA knockdown prevented development of the adipocyte-induced malignant phenotype. Specifically, inhibition of CD36 attenuated adipocyte-induced cholesterol and lipid droplet accumulation and reduced intracellular reactive oxygen species (ROS) content. Metabolic analysis suggested that CD36 plays an essential role in the bioenergetic adaptation of OvCa cells in the adipocyte-rich microenvironment and governs their metabolic plasticity. Furthermore, the absence of CD36 affected cellular processes that play a causal role in peritoneal dissemination, including adhesion, invasion, migration and anchorage independent growth. Intraperitoneal injection of CD36-deficient cells or treatment with an anti-CD36 monoclonal antibody reduced tumour burden in mouse xenografts. Moreover, a matched cohort of primary and metastatic human ovarian tumours showed upregulation of CD36 in the metastatic tissues, a finding confirmed in three public gene expression datasets. These results suggest that omental adipocytes reprogram tumour metabolism through the upregulation of CD36 in ovarian cancer cells. Targeting the stromal-tumour metabolic interface via CD36 inhibition may prove to be an effective treatment strategy against OvCa metastasis.

Author(s): Benitah, Salvador Aznar; Bracken, Adrian; Dou, Yali; Huangfu, Danwei; Ivanova, Natalia; Koseki, Haruhiko; Laurent, Louise; Lim, Daniel A; Meshorer, Eran; Pombo, Ana; Sander, Maike; Xu, Guo-Liang

Mammals rely on a network of circadian clocks to control daily systemic metabolism and physiology. The central pacemaker in the suprachiasmatic nucleus (SCN) is considered hierarchically dominant over peripheral clocks, whose degree of independence, or tissue-level autonomy, has never been ascertained invivo. Using arrhythmic Bmal1-null mice, we generated animals with reconstituted circadian expression of BMAL1 exclusively in the liver (Liver-RE). High-throughput transcriptomics and metabolomics show that the liver has independent circadian functions specific for metabolic processes such as the NAD+ salvage pathway and glycogen turnover. However, although BMAL1 occupies chromatin at most genomic targets in Liver-RE mice, circadian expression is restricted to ∼10% of normally rhythmic transcripts. Finally, rhythmic clock gene expression is lost in Liver-RE mice under constant darkness. Hence, full circadian function in the liver depends on signals emanating from other clocks, and light contributes to tissue-autonomous clock function.

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. Core support from the MRC and grants from MRCT, CRUK and the AICR financially supported the research in J.G's laboratory. J.G. is also supported by the EMBO Young Investigator Programme

The reactivation of the INK4-ARF locus, which is epigenetically repressed by Polycomb proteins in healthy cells, is a hallmark of senescence. One mechanism of reactivating Polycomb-silenced genes is mediated by the epigenetic factor ZRF1, which associates with ubiquitinated histone H2A. We show that cells undergoing senescence following oncogenic Ras expression have increased ZRF1 levels, and that this binds to the p15INK4b, ARF and p16INK4a promoters. Furthermore, ZRF1 depletion in oncogenic Ras-expressing cells restores proliferation by preventing Arf and p16Ink4a expression, consequently bypassing senescence. Thus, ZRF1 regulates the INK4-ARF locus during cellular proliferation and senescence, and alterations in ZRF1 may contribute to tumorigenesis.

The Polycomb repressive complex 1 (PRC1) is required for decisions of stem cell fate. In mouse embryonic stem cells (ESCs), two major variations of PRC1 complex, defined by the mutually exclusive presence of Cbx7 or RYBP, have been identified. Here, we show that although the genomic localization of the Cbx7- and RYBP-containing PRC1 complexes overlaps in certain genes, it can also be mutually exclusive. At the molecular level, Cbx7 is necessary for recruitment of Ring1B to chromatin, whereas RYBP enhances the PRC1 enzymatic activity. Genes occupied by RYBP show lower levels of Ring1B and H2AK119ub and are consequently more highly transcribed than those bound by Cbx7. At the functional level, we show that genes occupied by RYBP are primarily involved in the regulation of metabolism and cell-cycle progression, whereas those bound by Cbx7 predominantly control early-lineage commitment of ESCs. Altogether, our results indicate that different PRC1 subtypes establish a complex pattern of gene regulation that regulates common and nonoverlapping aspects of ESC pluripotency and differentiation. This work was supported by grants from the Spanish "Ministerio de/nEducación y Ciencia" (BFU2010-18692), from AGAUR, and from by European Commission's 7th Framework Program 4DCellFate grant number 277899 to L.D.C.; L.M. was supported by a postdoctoral CRG-Novartis fellowship

Mammalian embryonic development is a tightly regulated process that, from a single zygote, produces a large number of cell types with hugely divergent functions. Distinct cellular differentiation programmes are facilitated by tight transcriptional and epigenetic regulation. However, the contribution of epigenetic regulation to tissue homeostasis after the completion of development is less well understood. In this Review, we explore the effects of epigenetic dysregulation on adult stem cell function. We conclude that, depending on the tissue type and the epigenetic regulator affected, the consequences range from negligible to stem cell malfunction and disruption of tissue homeostasis, which may predispose to diseases such as cancer.

International audience; Pw1/Peg3 is a parentally imprinted gene expressed in adult stem cells in every tissue thus far examined including the stem cells of the hair follicle. Using a Pw1/Peg3 reporter mouse, we carried out a detailed dissection of the stem cells in the bulge, which is a major stem cell compartment of the hair follicle in mammalian skin. We observed that PW1/Peg3 expression initiates upon placode formation during fetal development, coincident with the establishment of the bulge stem cells. In the adult, we observed that PW1/Peg3 expression is found in both CD34+ and CD34- populations of bulge stem cells. We demonstrate that both populations can give rise to new hair follicles, reconstitute their niche, and self-renew. These results demonstrate that PW1/Peg3 is a reliable marker of the full population of follicle stem cells and reveal a novel CD34- bulge stem-cell population. Stem Cells 2017;35:1015–1027

Polycomb complex member Cbx4 represses nonepidermal lineage and cell cycle inhibitor genes in the epidermal keratinocytes and operates as a direct p63 target, maintaining epithelial identity and proliferative activity in the developing epidermis., During development, multipotent progenitor cells establish lineage-specific programmers of gene activation and silencing underlying their differentiation into specialized cell types. We show that the Polycomb component Cbx4 serves as a critical determinant that maintains the epithelial identity in the developing epidermis by repressing nonepidermal gene expression programs. Cbx4 ablation in mice results in a marked decrease of the epidermal thickness and keratinocyte (KC) proliferation associated with activation of numerous neuronal genes and genes encoding cyclin-dependent kinase inhibitors (p16/p19 and p57). Furthermore, the chromodomain- and SUMO E3 ligase–dependent Cbx4 activities differentially regulate proliferation, differentiation, and expression of nonepidermal genes in KCs. Finally, Cbx4 expression in KCs is directly regulated by p63 transcription factor, whereas Cbx4 overexpression is capable of partially rescuing the effects of p63 ablation on epidermal development. These data demonstrate that Cbx4 plays a crucial role in the p63-regulated program of epidermal differentiation, maintaining the epithelial identity and proliferative activity in KCs via repression of the selected nonepidermal lineage and cell cycle inhibitor genes.

One fundamental aspect of biological sciences is to understand how different cell fates are established during development and how cellular identity is maintained in adulthood. The molecular root of these processes is the interaction of chromatin modifications with epigenetic regulators and tissue-specific transcription factors. The concerted activities among them robustly define lineage specification, but also allow a degree of lineage flexibility required for tissue homeostasis and repair. The epidermis is emerging as an ideal model system to characterize the functional roles of epigenetic mechanisms that orchestrate organogenesis and adult tissue homeostasis. Here we summarize and discuss emerging roles of chromatin and epigenetic complexes in the mammalian epidermis.

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.

During aging, stromal functions are thought to be impaired, but little is known whether this stems from changes of fibroblasts. Using population- and single-cell transcriptomics, as well as long-term lineage tracing, we studied whether murine dermal fibroblasts are altered during physiological aging under different dietary regimes that affect longevity. We show that the identity of old fibroblasts becomes undefined, with the fibroblast states present in young skin no longer clearly demarcated. In addition, old fibroblasts not only reduce the expression of genes involved in the formation of the extracellular matrix, but also gain adipogenic traits, paradoxically becoming more similar to neonatal pro-adipogenic fibroblasts. These alterations are sensitive to systemic metabolic changes: long-term caloric restriction reversibly prevents them, whereas a high-fat diet potentiates them. Our results therefore highlight loss of cell identity and the acquisition of adipogenic traits as a mechanism underlying cellular aging, which is influenced by systemic metabolism. M.C.S. was supported by a Boehringer Ingelheim Fonds International PhD fellowship. IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from MINECO (Government of Spain). H.H. is a Miguel Servet (CP14/00229) researcher funded by the Spanish Institute of Health Carlos III (ISCIII). This work has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sk1odowska-Curie grant agreement No. H2020-MSCA-ITN-2015-675752 (SINGEK)

10 years ago it was reported that overexpression of the oncogene c-Myc in human epidermal stem cells stimulates differentiation rather than uncontrolled proliferation. This was, understandably, greeted with scepticism by researchers. However, subsequent studies have confirmed that Myc can stimulate epidermal stem cells to differentiate and shed light on the underlying mechanisms. Two cancer relevant concepts emerge. First, Myc regulates similar genes in different cell types, but the biological consequences are context-dependent. Second, Myc activation is not a simple ‘on/off’ switch. The cellular response depends on the strength and duration of Myc activity, which in turn is affected by the multiple cofactors and regulatory pathways with which Myc interacts.

The fact that the identity of the cells that initiate metastasis in most human cancers is unknown hampers the development of antimetastatic therapies. Here we describe a subpopulation of CD44bright cells in human oral carcinomas that do not overexpress mesenchymal genes, are slow-cycling, express high levels of the fatty acid receptor CD36 and lipid metabolism genes, and are unique in their ability to initiate metastasis. Palmitic acid or a high-fat diet specifically boosts the metastatic potential of CD36+ metastasis-initiating cells in a CD36-dependent manner. The use of neutralizing antibodies to block CD36 causes almost complete inhibition of metastasis in immunodeficient or immunocompetent orthotopic mouse models of human oral cancer, with no side effects. Clinically, the presence of CD36+ metastasis-initiating cells correlates with a poor prognosis for numerous types of carcinomas, and inhibition of CD36 also impairs metastasis, at least in human melanoma- and breast cancer-derived tumours. Together, our results indicate that metastasis-initiating cells particularly rely on dietary lipids to promote metastasis.

Myc plays a key role in homeostasis of the skin. We show that Miz1, which mediates Myc repression of gene expression, is expressed in the epidermal basal layer. A large percentage of genes regulated by the Myc–Miz1 complex in keratinocytes encode proteins involved in cell adhesion, and some, including the α6 and β1 integrins, are directly bound by Myc and Miz1 in vivo. Using a Myc mutant deficient in Miz1 binding (MycV394D), we show that Miz1 is required for the effects of Myc on keratinocyte responsiveness to TGF-β. Myc, but not MycV394D, decreases keratinocyte adhesion and spreading. In reconstituted epidermis, Myc induces differentiation and loss of cell polarization in a Miz1-dependent manner. In vivo, overexpression of β1 integrins restores basal layer polarity and prevents Myc-induced premature differentiation. Our data show that regulation of cell adhesion is a major function of the Myc–Miz1 complex and suggest that it may contribute to Myc-induced exit from the epidermal stem cell compartment.

Rho proteins belong to the small GTPases superfamily. They function as molecular switches that, in response to diverse stimuli, control key signaling and structural aspects of the cell. Although early studies proposed a role for Rho GTPases in cellular transformation, this effect was underestimated due to the fact that no genetic mutations affecting Rho-encoding genes were found in tumors. Recently, it has become evident that Rho GTPases participate in the carcinogenic process by either overexpression of some of the members of the family with oncogenic activity, downmodulation of other members with suggested tumor suppressor activity, or by alteration of upstream modulators or downstream effectors. Thus, alteration of the levels of expression of different members of the family of Rho GTPases has been detected in many types of human tumors leading to a great interest in the cellular effects elicited by these oncoproteins. This essay reviews the current evidence of dysregulation of Rho signaling by overexpression in human tumors.

Rho GTPases are overexpressed in a variety of human tumors contributing to both tumor proliferation and metastasis. Recently, several studies demonstrate an essential role of transcriptional regulation in Rho GTPases-induced oncogenesis. Herein, we demonstrate that RhoA, Rac1, and Cdc42 promote the expression of cyclooxygenase-2 (COX-2) at the transcriptional level by a mechanism that is dependent on the transcription factor nuclear factor-kappaB (NF-kappaB), but not Stat3, a transcription factor required for RhoA-induced tumorigenesis. With respect to RhoA, this effect is dependent on ROCK, but not PKN. Treatment of RhoA-, Rac1-, and Cdc42-transformed epithelial cells with Sulindac and NS-398, two well-characterized nonsteroid antiinflammatory drugs (NSAIDs), results in growth inhibition as determined by cell proliferation assays. Accordingly, tumor growth of RhoA-expressing epithelial cells in syngeneic mice is strongly inhibited by NS-398 treatment. The effect of NSAIDs over RhoA-induced tumor growth is not exclusively dependent on COX-2 because DNA-binding of NF-kappaB is also abolished upon NSAIDs treatment, resulting in complete loss of COX-2 expression. Finally, treatment of RhoA-transformed cells with Bay11-7083, a specific NF-kappaB inhibitor, leads to inhibition of cell proliferation. We suggest that treatment of human tumors that overexpress Rho GTPases with NSAIDs and drugs that target NF-kappaB could constitute a valid antitumoral strategy.

Chemotherapeutic agents such as cisplatin induce persistent activation of N-terminal c-Jun Kinase, which in turn mediates induction of apoptosis. By using a common MAPK Kinase, MEKK1, cisplatin also activates the survival transcription factor NFκB. We have found a cross-talk between c-Jun expression and NFκB transcriptional activation in response to cisplatin. Fibroblast derived from c-jun knock out mice are more resistant to cisplatin-induced cell death, and this survival advantage is mediated by upregulation of NFκB-dependent transcription and expression of MIAP3. This process can be reverted by ectopic expression of c-Jun in c-jun−/−fibroblasts, which decreases p65 transcriptional activity back to normal levels. Negative regulation of NFκB-dependent transcription by c-jun contributes to cisplatin-induced cell death, which suggests that inhibition of NFκB may potentiate the antineoplastic effect of conventional chemotherapeutic agents.

The process of aging and circadian rhythms are intimately intertwined, but how peripheral clocks involved in metabolic homeostasis contribute to aging remains unknown. Importantly, caloric restriction (CR) extends lifespan in several organisms and rewires circadian metabolism. Using young versus old mice, fed ad libitum or under CR, we reveal reprogramming of the circadian transcriptome in the liver. These age-dependent changes occur in a highly tissue-specific manner, as demonstrated by comparing circadian gene expression in the liver versus epidermal and skeletal muscle stem cells. Moreover, de novo oscillating genes under CR show an enrichment in SIRT1 targets in the liver. This is accompanied by distinct circadian hepatic signatures in NAD(+)-related metabolites and cyclic global protein acetylation. Strikingly, this oscillation in acetylation is absent in old mice while CR robustly rescues global protein acetylation. Our findings indicate that the clock operates at the crossroad between protein acetylation, liver metabolism and aging.

Mammalian epidermis is maintained by self-renewal of stem cells, but the underlying mechanisms are unknown. Deletion of Rac1, a Rho guanosine triphosphatase, in adult mouse epidermis stimulated stem cells to divide and undergo terminal differentiation, leading to failure to maintain the interfollicular epidermis, hair follicles, and sebaceous glands. Rac1 exerts its effects in the epidermis by negatively regulating c-Myc through p21-activated kinase 2 (PAK2) phosphorylation. We conclude that a pleiotropic regulator of cell adhesion and the cytoskeleton plays a critical role in controlling exit from the stem cell niche and propose that Rac and Myc represent a global stem cell regulatory axis.

In the past years, our view of the molecular and cellular mechanisms that ensure the self-renewal of the skin has dramatically changed. Several populations of stem cells have been identified that differ in their spatio-temporal contribution to their compartment in steady-state and damaged conditions, suggesting that epidermal stem cell heterogeneity is far greater than previously anticipated. There is also increasing evidence that these different stem cells require a tightly controlled spatial and temporal communication between other skin residents to carry out their function.

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. This work has been funded by an ERC Consolidator Grant (STEMCLOCK) to S.A.B. and by the PROSPECTS, grant agreement number HEALTH-F4-2008-201648, to L.S. from the European Union. G.S. is funded by an AXA Fund fellowship. K.T. is funded by a La Caixa Ph.D. fellowship./nResearch in the lab of B.L. is funded by the European Research Council (ERC), MINECO Plan Nacional grant BFU2011-26206, ERASysBio+ ERANET project EUI2009-04059 GRAPPLE, the EMBO Young Investigator Program, EU Framework 7 project 277899 4DCellFate, and the EMBL/CRG Systems Biology Program

Polycomb group proteins (PcGs) generate chromatin-modifying complexes that regulate gene expression. PcGs are categorized into two major groups, polycomb repressive complex 1 (PRC1) and 2 (PRC2), which have classically been thought to function together. Here we discuss recent data challenging this model indicating that the distinct subunit composition of PRC1 confers specific and nonoverlapping functions in embryonic and adult stem cells.

One of the most striking epigenetic alterations that occurs at the level of the nucleosome is the complete exchange of the canonical H2A histones for the macroH2A variant. Here, we provide insight into the poorly recognized function of macroH2A in transcriptional activation and demonstrate its relevance in embryonic and adult stem cells. Knockdown of macroH2A1 in mouse embryonic stem (mES) cells limited their capacity to differentiate but not their self-renewal. The loss of macroH2A1 interfered with the proper activation of differentiation genes, most of which are direct target genes of macroH2A. Additionally, macroH2A1-deficient mES cells displayed incomplete inactivation of pluripotency genes and formed defective embryoid bodies. In vivo, macroH2A1-deficient teratomas contained a massive expansion of malignant, undifferentiated carcinoma tissue. In the heterogeneous culture of primary human keratinocytes, macroH2A1 levels negatively correlated with the self-renewal capacity of the pluripotent compartment. Together these results establish macroH2A1 as a critical chromatin component that regulates the delicate balance between self-renewal and differentiation of embryonic and adult stem cells.

The thymic stroma supports T lymphocyte development and consists of an epithelium maintained by thymic epithelial progenitors. The molecular pathways that govern epithelial homeostasis are poorly understood. Here we demonstrate that deletion of Rac1 in Keratin 5/Keratin 14 expressing embryonic and adult thymic epithelial cells leads to loss of the thymic epithelial compartment. Rac1 deletion led to an increase in c-Myc expression and a generalized increase in apoptosis associated with a decrease in thymic epithelial proliferation. Our results suggest Rac1 maintains the epithelial population, and equilibrium between Rac1 and c-Myc may control proliferation, apoptosis and maturation of the thymic epithelial compartment. Understanding thymic epithelial maintenance is a step toward the dual goals of in vitro thymic epithelial cell culture and T cell differentiation, and the clinical repair of thymic damage from graft-versus-host-disease, chemotherapy or irradiation.

During epidermal chemical carcinogenesis benign papillomas convert to squamous cell carcinomas, some of which undergo epithelial-mesenchymal conversion to highly malignant spindle cell tumors. TGFbeta inhibits early stages of carcinogenesis but promotes the spindle cell phenotype in later stages. One hallmark of spindle cell tumors is upregulation of the alpha 5 beta 1 integrin fibronectin receptor. To examine the significance of altered alpha 5 beta1 integrin expression, we induced tumors in transgenic mice expressing alpha 5 beta1 in the suprabasal epidermal layers. Invalpha 5 beta1 mice developed threefold more papillomas and squamous cell carcinomas than wild-type (Wt) littermates; however, no spindle cell tumors or increased metastases were observed. Suprabasal expression of the alpha 6 beta 4 integrin increases squamous cell carcinoma formation and decreases TGFbeta sensitivity while alpha 3 beta1 may have the opposite effect. In contrast, nuclear phosphoSmad2 labeling in Invalpha 5 beta1 epidermis and tumors was indistinguishable from Wt, and suprabasal alpha 5 beta1 did not block TGFbeta-induced Smad2/3 translocation or growth inhibition in cultured keratinocytes. We conclude that upregulation of alpha 5 beta1 does not predispose the epidermis to undergo conversion to spindle cell tumors and that the mechanism by which alpha 5 beta1 influences susceptibility to carcinogenesis is independent of perturbed TGFbeta signaling.

Homeostasis of most adult tissues is maintained by balancing stem cell self-renewal and differentiation, but whether post-transcriptional mechanisms can regulate this process is unknown. Here, we identify that an RNA methyltransferase (Misu/Nsun2) is required to balance stem cell self-renewal and differentiation in skin. In the epidermis, this methyltransferase is found in a defined sub-population of hair follicle stem cells poised to undergo lineage commitment, and its depletion results in enhanced quiescence and aberrant stem cell differentiation. Our results reveal that post-transcriptional RNA methylation can play a previously unappreciated role in controlling stem cell fate., Author Summary We demonstrate that the RNA methyltransferase activity of Misu/NSun2 is required for the proper maintenance of the epidermal differentiation program, and thus post-transcriptional mechanisms are involved in controlling the balance between stem cell self-renewal and differentiation.

Tissue-specific stem cells sustain organs for a lifetime through self-renewal and generating differentiated progeny. Although tissue stem cells are established during organogenesis, the precise origin of most adult stem cells in the developing embryo is unclear. Mammalian skin is one of the best-studied epithelial systems containing stem cells to date, however the origin of most of the stem cell populations found in the adult epidermis is unknown. Here, we try to recapitulate the emergence and genesis of an ectodermal stem cell during development until the formation of an adult skin. We ask whether skin stem cells share key transcriptional regulators with their embryonic counterparts and discuss whether embryonic-like stem cells may persist through to adulthood in vivo.