IL-1α cleavage by inflammatory caspases of the noncanonical inflammasome controls the senescence-associated secretory phenotype.

Interleukin-1 alpha (IL-1α) is a powerful cytokine that modulates immunity, and requires canonical cleavage by calpain for full activity. Mature IL-1α is produced after inflammasome activation and during cell senescence, but the protease cleaving IL-1α in these contexts is unknown. We show IL-1α is activated by caspase-5 or caspase-11 cleavage at a conserved site. Caspase-5 drives cleaved IL-1α release after human macrophage inflammasome activation, while IL-1α secretion from murine macrophages only requires caspase-11, with IL-1β release needing caspase-11 and caspase-1. Importantly, senescent human cells require caspase-5 for the IL-1α-dependent senescence-associated secretory phenotype (SASP) in vitro, while senescent mouse hepatocytes need caspase-11 for the SASP-driven immune surveillance of senescent cells in vivo. Together, we identify IL-1α as a novel substrate of noncanonical inflammatory caspases and finally provide a mechanism for how IL-1α is activated during senescence. Thus, targeting caspase-5 may reduce inflammation and limit the deleterious effects of accumulated senescent cells during disease and Aging.Work was funded by British Heart Foundation grants FS/13/3/30038, FS/18/19/33371 and RG/16/8/32388 (MC); Cancer Research UK Cambridge Institute Core Grant C14303/A17197, Medical Research Council grants MR/M013049/1 and MR/R010013/1 (MN); and the Cambridge NIHR Biomedical Research Centre

NOTCH-mediated non-cell autonomous regulation of chromatin structure during senescence.

Senescent cells interact with the surrounding microenvironment achieving diverse functional outcomes. We have recently identified that NOTCH1 can drive 'lateral induction' of a unique senescence phenotype in adjacent cells by specifically upregulating the NOTCH ligand JAG1. Here we show that NOTCH signalling can modulate chromatin structure autonomously and non-autonomously. In addition to senescence-associated heterochromatic foci (SAHF), oncogenic RAS-induced senescent (RIS) cells exhibit a massive increase in chromatin accessibility. NOTCH signalling suppresses SAHF and increased chromatin accessibility in this context. Strikingly, NOTCH-induced senescent cells, or cancer cells with high JAG1 expression, drive similar chromatin architectural changes in adjacent cells through cell-cell contact. Mechanistically, we show that NOTCH signalling represses the chromatin architectural protein HMGA1, an association found in multiple human cancers. Thus, HMGA1 is involved not only in SAHFs but also in RIS-driven chromatin accessibility. In conclusion, this study identifies that the JAG1-NOTCH-HMGA1 axis mediates the juxtacrine regulation of chromatin architecture

Developmental Programming Mediated by Complementary Roles of Imprinted Grb10 in Mother and Pup

Developmental programming links growth in early life with health status in adulthood. Although environmental factors such as maternal diet can influence the growth and adult health status of offspring, the genetic influences on this process are poorly understood. Using the mouse as a model, we identify the imprinted gene Grb10 as a mediator of nutrient supply and demand in the postnatal period. The combined actions of Grb10 expressed in the mother, controlling supply, and Grb10 expressed in the offspring, controlling demand, jointly regulate offspring growth. Furthermore, Grb10 determines the proportions of lean and fat tissue during development, thereby influencing energy homeostasis in the adult. Most strikingly, we show that the development of normal lean/fat proportions depends on the combined effects of Grb10 expressed in the mother, which has the greater effect on offspring adiposity, and Grb10 expressed in the offspring, which influences lean mass. These distinct functions of Grb10 in mother and pup act complementarily, which is consistent with a coadaptation model of imprinting evolution, a model predicted but for which there is limited experimental evidence. In addition, our findings identify Grb10 as a key genetic component of developmental programming, and highlight the need for a better understanding of mother-offspring interactions at the genetic level in predicting adult disease risk

G-quadruplex structures mark human regulatory chromatin

G-quadruplex (G4) structural motifs have been linked to transcription, replication and genome instability and are implicated in cancer and other diseases. However, it is crucial to demonstrate the bona fide formation of G4 structures within an endogenous chromatin context. Herein we address this through the development of G4 ChIP-seq, an antibody-based G4 chromatin immunoprecipitation and high-throughput sequencing approach. We find ∼10,000 G4 structures in human chromatin, predominantly in regulatory, nucleosome-depleted regions. G4 structures are enriched in the promoters and 5' UTRs of highly transcribed genes, particularly in genes related to cancer and in somatic copy number amplifications, such as $\textit{MYC}$. Strikingly, $\textit{de novo}$ and enhanced G4 formation are associated with increased transcriptional activity, as shown by HDAC inhibitor-induced chromatin relaxation and observed in immortalized as compared to normal cellular states. Our findings show that regulatory, nucleosome-depleted chromatin and elevated transcription shape the endogenous human G4 DNA landscape.European Molecular Biology Organization (EMBO Long-Term Fellowship), University of Cambridge, Cancer Research UK (Grant ID: C14303/A17197), Wellcome Trust (Grant ID: 099232/z/12/z

NOTCH1 mediates a switch between two distinct secretomes during senescence

Senescence, a persistent form of cell-cycle arrest, is often associated with a diverse secretome, which provides complex functionality for senescent cells within the tissue microenvironment. We show that oncogene-induced senescence is accompanied by a dynamic fluctuation of NOTCH1 activity, which drives a TGF-β-rich secretome, while suppressing the senescence-associated pro-inflammatory secretome through inhibition of C/EBPβ. NOTCH1 and NOTCH1-driven TGF-β contribute to 'lateral induction of senescence' through a juxtacrine NOTCH-JAG1 pathway. In addition, NOTCH1 inhibition during senescence facilitates upregulation of pro-inflammatory cytokines, promoting lymphocyte recruitment and senescence surveillance in vivo. As enforced activation of NOTCH1 signalling confers a near mutually exclusive secretory profile compared with typical senescence, our data collectively indicate that the dynamic alteration of NOTCH1 activity during senescence dictates a functional balance between these two distinct secretomes: one representing TGF-β and the other pro-inflammatory cytokines, highlighting that NOTCH1 is a temporospatial controller of secretome composition.This work was supported by the University of Cambridge, Cancer Research UK and Hutchison Whampoa. The M.N. laboratory is supported by Cancer Research UK Cambridge Institute Core Grant (C14303/A17197). M.H. was supported by CRUK Translational Medicine Research Fellowship and CRUK Clinician Scientist Fellowship (C52489/A19924). This work was also supported by a Wellcome Trust PRF (WT101835) to P.J.L., a Wellcome Trust Senior Fellowship to M.P.W. (108070/Z/15/Z), a Wellcome Trust Training Fellowship to N.J.M. (093964/Z/10/Z), and a Wellcome Trust Intermediate Fellowship (097162/Z/11/Z) to S.S. L.Z. was funded by the German Research Foundation (DFG; grants FOR2314 and SFB685), the Gottfried Wilhelm Leibniz Program, the European Research Council (projects ‘CholangioConcept’), the German Ministry for Education and Research (BMBF) (eMed-Multiscale HCC), the German Universities Excellence Initiative (third funding line: ‘future concept’), the German Center for Translational Cancer Research (DKTK) and the German–Israeli Cooperation in Cancer Research (DKFZ–MOST).This is the author accepted manuscript. The final version is available from Nature Publishing Group at http://dx.doi.org/10.1038/ncb3397

Core-Shell Spheroid-Laden Microgels Crosslinked under Biocompatible Conditions for Probing Cancer-Stromal Communication

Funder: H2020 European Research Council; Id: http://dx.doi.org/10.13039/100010663Funder: China ScholarshipFunder: College of Engineering, University of Canterbury; Id: http://dx.doi.org/10.13039/100014447Multicellular cancer spheroids (MCSs) have emerged as a promising in vitro model that recaptures many features of solid tumours in vivo. To generate cancer spheroids, cells are encapsulated in microgels with high throughput. While the biophysical properties of the cancer spheroid and biomaterial influence the function and behavior of the cells, characterization of these properties remains largely unexplored. In addition, many existing techniques lack control over cell positioning, resulting in the formation of spheroids with large variability. Herein, a versatile, microfluidic platform for generating biocompatible core–shell microgels with uniform cancer spheroids is reported. In addition, an in situ micromechanics measuring device to determine the stiffness of individual artificial cancer niches is used. The power of the microfluidics‐based method by making MCS‐laden microgels to model the interactions of stromal cancer cells is demonstrated. Together with in vivo data, it is shown that tumor cell proliferation is promoted by cocultured fibroblasts

Transcription-dependent cohesin repositioning rewires chromatin loops in cellular senescence

AbstractSenescence is a state of stable proliferative arrest, generally accompanied by the senescence-associated secretory phenotype, which modulates tissue homeostasis. Enhancer-promoter interactions, facilitated by chromatin loops, play a key role in gene regulation but their relevance in senescence remains elusive. Here, we use Hi-C to show that oncogenic RAS-induced senescence in human diploid fibroblasts is accompanied by extensive enhancer-promoter rewiring, which is closely connected with dynamic cohesin binding to the genome. We find de novo cohesin peaks often at the 3′ end of a subset of active genes. RAS-induced de novo cohesin peaks are transcription-dependent and enriched for senescence-associated genes, exemplified by IL1B, where de novo cohesin binding is involved in new loop formation. Similar IL1B induction with de novo cohesin appearance and new loop formation are observed in terminally differentiated macrophages, but not TNFα-treated cells. These results suggest that RAS-induced senescence represents a cell fate determination-like process characterised by a unique gene expression profile and 3D genome folding signature, mediated in part through cohesin redistribution on chromatin.</jats:p

Locus-specific induction of gene expression from heterochromatin loci during cellular senescence

Senescence is a fate-determined state, accompanied by reorganization of heterochromatin. Although lineage-appropriate genes can be temporarily repressed through facultative heterochromatin, stable silencing of lineage-inappropriate genes often involves the constitutive heterochromatic mark, histone H3 lysine 9 trimethylation (H3K9me3). The fate of these heterochromatic genes during senescence is unclear. In the present study, we show that a small number of lineage-inappropriate genes, exemplified by the LCE2 skin genes, are derepressed during senescence from H3K9me3 regions in fibroblasts. DNA FISH experiments reveal that these gene loci, which are condensed at the nuclear periphery in proliferative cells, are decompacted during senescence. Decompaction of the locus is not sufficient for LCE2 expression, which requires p53 and C/EBPβ signaling. NLRP3, which is predominantly expressed in macrophages from an open topologically associated domain (TAD), is also derepressed in senescent fibroblasts due to the local disruption of the H3K9me3-rich TAD that contains it. NLRP3 has been implicated in the amplification of inflammatory cytokine signaling in senescence and aging, highlighting the functional relevance of gene induction from ‘permissive’ H3K9me3 regions in senescent cells

Comparison of <i>Grb10KO</i> and <i>Grb10Δ2-4</i> mice.

<p>(A) Structure of <i>Grb10</i>, according to UCSC annotation, showing numbered exons (boxes) and translated regions (filled boxes). The integrated gene-trap cassettes include splice acceptor (SA) and polyadenylation (pA) signals, and a <i>LacZ</i> reporter. (B) Comparative <i>LacZ</i> staining of bisected embryos at e14.5 inheriting the <i>Grb10KO</i> and <i>Grb10Δ2-4</i> alleles through each of the parental lines. CNS expression observed in <i>Grb10KO</i>^+/p embryos is not detected in <i>Grb10Δ2-4</i>^+/p embryos. (C) Comparative <i>LacZ</i> staining of adult mammary glands at days 7.5 and 12.5 of gestation (G7.5, G12.5) and day 6 of lactation (L6), showing pregnancy-dependent reporter expression in <i>Grb10KO</i>^m/+ but not <i>Grb10Δ2-4</i>^m/+ females. WT (+/+) glands were stained with carmine alum to illustrate morphological changes.</p

Characterisation of CRM1 and STAT5-mediated expression of <i>Grb10</i>.

<p>(A) <i>In silico</i> identification of conserved elements among selected vertebrate sequences. Conserved intronic sequences between <i>Grb10</i> homologs are plotted against annotated mouse transcripts. The PReMod track shows the position of the single regulatory module (CRM1). This site aligns with a sequence highly conserved between mouse, human, chimpanzee, cow, and chicken (highlighted). (B) Assay for DNase I hypersensitivity at CRM1, using probe A. A 6 kb <i>Bam</i>HI fragment was detected in all samples. A 3.8 kb DNase I digestion fragment was detected in brain, but not liver, chromatin exposed to 200 U DNase I (arrow). The label “B” indicates a <i>Bam</i>HI site. (C) <i>In situ</i> hybridisation autoradiographs showing examples of overlapping sites of <i>Grb10</i> and <i>Stat5b</i> mRNA expression in adult mouse brain, including the arcuate nucleus of the hypothalamus (ARC), dorsomedial nucleus of the hypothalamus (DMH), lateral septal nucleus (LSV), medial amygdaloid nucleus (posteroventral part) (MePV), medial habenular nucleus (MHb), medial preoptic nucleus (MPA), median preoptic nucleus (medial part) (MPOM), periaqueductal grey (PAG), paraventricular thalamic nucleus (PVA), paraventricular nucleus of the hypothalamus (PVH), supraoptic nucleus (SON), ventromedial nucleus of the hypothalamus (VMH), and ventraltegmental area (VTA). (D) <i>In vitro</i> transfection assay of the enhancer capability of CRM1. Luciferase activity was measured in cells transfected with a minimal promoter driving luciferase (pGL3-Pro) or with CRM1 cloned upstream of the minimal promoter (pGL3-Pro-CRM1). Only pGL3-Pro-CRM1 responded to increasing doses of constitutively active STAT5b (STAT5b1*6). ***<i>p</i><0.001 (one-way ANOVA).</p