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• W2799540870 abstract "•HMGB2 nuclear depletion is an early event on the path to senescence•HMGB2 binds positions both at TAD boundaries and within TADs•The loss of HMGB2 induces heterochromatic and transcriptional changes•HMGB2 suffices for instructing and rescuing senescence-induced CTCF clustering Processes like cellular senescence are characterized by complex events giving rise to heterogeneous cell populations. However, the early molecular events driving this cascade remain elusive. We hypothesized that senescence entry is triggered by an early disruption of the cells’ three-dimensional (3D) genome organization. To test this, we combined Hi-C, single-cell and population transcriptomics, imaging, and in silico modeling of three distinct cells types entering senescence. Genes involved in DNA conformation maintenance are suppressed upon senescence entry across all cell types. We show that nuclear depletion of the abundant HMGB2 protein occurs early on the path to senescence and coincides with the dramatic spatial clustering of CTCF. Knocking down HMGB2 suffices for senescence-induced CTCF clustering and for loop reshuffling, while ectopically expressing HMGB2 rescues these effects. Our data suggest that HMGB2-mediated genomic reorganization constitutes a primer for the ensuing senescent program. Processes like cellular senescence are characterized by complex events giving rise to heterogeneous cell populations. However, the early molecular events driving this cascade remain elusive. We hypothesized that senescence entry is triggered by an early disruption of the cells’ three-dimensional (3D) genome organization. To test this, we combined Hi-C, single-cell and population transcriptomics, imaging, and in silico modeling of three distinct cells types entering senescence. Genes involved in DNA conformation maintenance are suppressed upon senescence entry across all cell types. We show that nuclear depletion of the abundant HMGB2 protein occurs early on the path to senescence and coincides with the dramatic spatial clustering of CTCF. Knocking down HMGB2 suffices for senescence-induced CTCF clustering and for loop reshuffling, while ectopically expressing HMGB2 rescues these effects. Our data suggest that HMGB2-mediated genomic reorganization constitutes a primer for the ensuing senescent program. Since the original description of in vitro replicative senescence (Hayflick, 1965Hayflick L. The limited in vitro lifetime of human diploid cell strains.Exp. Cell Res. 1965; 37: 614-636Crossref PubMed Scopus (4284) Google Scholar), in vivo implications in development, wound healing, organismal aging, and disease have been uncovered (van Deursen, 2014van Deursen J.M. The role of senescent cells in ageing.Nature. 2014; 509: 439-446Crossref PubMed Scopus (1446) Google Scholar). In addition, clearance of senescent cells in mice was shown to improve health- and lifespan (de Keizer, 2017de Keizer P.L. The fountain of youth by targeting senescent cells?.Trends Mol. Med. 2017; 23: 6-17Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Senescence entry is a result of integrated autocrine and paracrine signaling in the population (Acosta et al., 2013Acosta J.C. Banito A. Wuestefeld T. Georgilis A. Janich P. Morton J.P. Athineos D. Kang T.W. Lasitschka F. Andrulis M. et al.A complex secretory program orchestrated by the inflammasome controls paracrine senescence.Nat. Cell Biol. 2013; 15: 978-990Crossref PubMed Scopus (1170) Google Scholar, Davalos et al., 2013Davalos A.R. Kawahara M. Malhotra G.K. Schaum N. Huang J. Ved U. Beausejour C.M. Coppe J.P. Rodier F. Campisi J. p53-dependent release of Alarmin HMGB1 is a central mediator of senescent phenotypes.J. Cell Biol. 2013; 201: 613-629Crossref PubMed Scopus (282) Google Scholar, Hoare and Narita, 2017Hoare M. Narita M. NOTCH and the 2 SASPs of senescence.Cell Cycle. 2017; 16: 239-240Crossref PubMed Scopus (11) Google Scholar) triggering replicative arrest, gene expression changes, secretory activity, and chromatin reorganization (Rai and Adams, 2013Rai T.S. Adams P.D. Lessons from senescence: chromatin maintenance in non-proliferating cells.Biochim. Biophys. Acta. 2013; 1819: 322-331Crossref PubMed Scopus (50) Google Scholar). Telomere shortening (Herbig et al., 2004Herbig U. Jobling W.A. Chen B.P. Chen D.J. Sedivy J.M. Telomere shortening triggers senescence of human cells through a pathway involving ATM, p53, and p21(CIP1), but not p16(INK4a).Mol. Cell. 2004; 14: 501-513Abstract Full Text Full Text PDF PubMed Scopus (956) Google Scholar), reorganization of heterochromatin and the lamina (Narita et al., 2006Narita M. Narita M. Krizhanovsky V. Nuñez S. Chicas A. Hearn S.A. Myers M.P. Lowe S.W. A novel role for high-mobility group a proteins in cellular senescence and heterochromatin formation.Cell. 2006; 126: 503-514Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar, Sadaie et al., 2013Sadaie M. Salama R. Carroll T. Tomimatsu K. Chandra T. Young A.R. Narita M. Pérez-Mancera P.A. Bennett D.C. Chong H. et al.Redistribution of the Lamin B1 genomic binding profile affects rearrangement of heterochromatic domains and SAHF formation during senescence.Genes Dev. 2013; 27: 1800-1808Crossref PubMed Scopus (196) Google Scholar, Shah et al., 2013Shah P.P. Donahue G. Otte G.L. Capell B.C. Nelson D.M. Cao K. Aggarwala V. Cruickshanks H.A. Rai T.S. McBryan T. et al.Lamin B1 depletion in senescent cells triggers large-scale changes in gene expression and the chromatin landscape.Genes Dev. 2013; 27: 1787-1799Crossref PubMed Scopus (353) Google Scholar, Swanson et al., 2013Swanson E.C. Manning B. Zhang H. Lawrence J.B. Higher-order unfolding of satellite heterochromatin is a consistent and early event in cell senescence.J. Cell Biol. 2013; 203: 929-942Crossref PubMed Scopus (153) Google Scholar, Zhang et al., 2005Zhang R. Poustovoitov M.V. Ye X. Santos H.A. Chen W. Daganzo S.M. Erzberger J.P. Serebriiskii I.G. Canutescu A.A. Dunbrack R.L. et al.Formation of MacroH2A-containing senescence-associated heterochromatin foci and senescence driven by ASF1a and HIRA.Dev. Cell. 2005; 8: 19-30Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar), activation of transposable elements (De Cecco et al., 2013De Cecco M. Criscione S.W. Peckham E.J. Hillenmeyer S. Hamm E.A. Manivannan J. Peterson A.L. Kreiling J.A. Neretti N. Sedivy J.M. Genomes of replicatively senescent cells undergo global epigenetic changes leading to gene silencing and activation of transposable elements.Aging Cell. 2013; 12: 247-256Crossref PubMed Scopus (268) Google Scholar), or epigenetic changes on histones and the primary DNA sequence have been observed (Cruickshanks et al., 2013Cruickshanks H.A. McBryan T. Nelson D.M. Vanderkraats N.D. Shah P.P. van Tuyn J. Singh Rai T. Brock C. Donahue G. Dunican D.S. et al.Senescent cells harbour features of the cancer epigenome.Nat. Cell Biol. 2013; 15: 1495-1506Crossref PubMed Scopus (230) Google Scholar, Franzen et al., 2017Franzen J. Zirkel A. Blake J. Rath B. Benes V. Papantonis A. Wagner W. Senescence-associated DNA methylation is stochastically acquired in subpopulations of mesenchymal stem cells.Aging Cell. 2017; 16: 183-191Crossref PubMed Scopus (49) Google Scholar, Neyret-Kahn et al., 2013Neyret-Kahn H. Benhamed M. Ye T. Le Gras S. Cossec J.C. Lapaquette P. Bischof O. Ouspenskaia M. Dasso M. Seeler J. et al.Sumoylation at chromatin governs coordinated repression of a transcriptional program essential for cell growth and proliferation.Genome Res. 2013; 23: 1563-1579Crossref PubMed Scopus (95) Google Scholar). Still, despite some of these events sufficing for replicative arrest, early events signaling senescence entry remain elusive. Thus, senescence is an attractive model for studying the structure-to-function relationship of chromosomes. This is now feasible due to the advent of Hi-C technology that captures spatial interactions within and between chromosomes (Belton et al., 2012Belton J.M. McCord R.P. Gibcus J.H. Naumova N. Zhan Y. Dekker J. Hi-C: a comprehensive technique to capture the conformation of genomes.Methods. 2012; 58: 268-276Crossref PubMed Scopus (501) Google Scholar). As a result of Hi-C studies, we now understand that chromosomes are partitioned into active A and inactive B compartments at the Mbp scale, and into consecutive topologically associating domains (TADs) at the sub-Mbp scale. TADs harbor chromatin loops that tend to interact with one another more frequently than with loops in other TADs (Dixon et al., 2012Dixon J.R. Selvaraj S. Yue F. Kim A. Li Y. Shen Y. Hu M. Liu J.S. Ren B. Topological domains in mammalian genomes identified by analysis of chromatin interactions.Nature. 2012; 485: 376-380Crossref PubMed Scopus (3981) Google Scholar). This higher-order organization is tightly linked to gene expression regulation, but also to cell-cycle progression (Naumova et al., 2013Naumova N. Imakaev M. Fudenberg G. Zhan Y. Lajoie B.R. Mirny L.A. Dekker J. Organization of the mitotic chromosome.Science. 2013; 342: 948-953Crossref PubMed Scopus (599) Google Scholar). For senescence, Hi-C has only been applied to fibroblasts undergoing oncogene-induced senescence (OIS) or maintained long term in “deep” senescence, both constituting end-point states. In OIS, interaction loss within heterochromatin and lamin-associated regions was observed, and in a next step clustering of heterochromatic stretches to form senescence-associated heterochromatic foci (SAHFs; Chandra et al., 2015Chandra T. Ewels P.A. Schoenfelder S. Furlan-Magaril M. Wingett S.W. Kirschner K. Thuret J.Y. Andrews S. Fraser P. Reik W. Global reorganization of the nuclear landscape in senescent cells.Cell Rep. 2015; 10: 471-483Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). In “deep” senescence, shorter-range interactions are favored over longer-range ones, thus compacting chromosome arms, while centromeres decondense (Criscione et al., 2016Criscione S.W. De Cecco M. Siranosian B. Zhang Y. Kreiling J.A. Sedivy J.M. Neretti N. Reorganization of chromosome architecture in replicative cellular senescence.Sci. Adv. 2016; 2: e1500882Crossref PubMed Scopus (81) Google Scholar). Therefore, whether spatial genome reorganization triggers replicative senescence entry remains unaddressed. We used three human primary cell types from individual donors—umbilical vein endothelial cells (HUVECs), fetal lung fibroblasts (IMR90s), and mesenchymal stromal cells (MSCs)—to identify a shared regulatory backbone instructing replicative arrest. We combined genomics, super-resolution microscopy, and single-cell sequencing to discover that proteins of the high-mobility group B (HMGB) family are implicated in regulating specific TAD boundaries, as well as in the spatial clustering of CTCF-bound chromatin. HMGBs are abundant nuclear proteins with characteristic HMG-box DNA-binding domains; they are known to distort DNA via unwinding, bending, or looping (Stros, 2010Stros M. HMGB proteins: interactions with DNA and chromatin.Biochim. Biophys. Acta. 2010; 1799: 101-113Crossref PubMed Scopus (433) Google Scholar). This renders them important for transcription, nucleosome remodeling, genome integrity, and recombination (Bonaldi et al., 2002Bonaldi T. Längst G. Strohner R. Becker P.B. Bianchi M.E. The DNA chaperone HMGB1 facilitates ACF/CHRAC-dependent nucleosome sliding.EMBO J. 2002; 21: 6865-6873Crossref PubMed Scopus (189) Google Scholar, Laurent et al., 2010Laurent B. Randrianarison-Huetz V. Maréchal V. Mayeux P. Dusanter-Fourt I. Duménil D. High-mobility group protein HMGB2 regulates human erythroid differentiation through trans-activation of GFI1B transcription.Blood. 2010; 115: 687-695Crossref PubMed Scopus (30) Google Scholar, Redmond et al., 2015Redmond A.M. Byrne C. Bane F.T. Brown G.D. Tibbitts P. O’Brien K. Hill A.D. Carroll J.S. Young L.S. Genomic interaction between ER and HMGB2 identifies DDX18 as a novel driver of endocrine resistance in breast cancer cells.Oncogene. 2015; 34: 3871-3880Crossref PubMed Scopus (22) Google Scholar, Lee et al., 2010Lee D. Kwon J.H. Kim E.H. Kim E.S. Choi K.Y. HMGB2 stabilizes p53 by interfering with E6/E6AP-mediated p53 degradation in human papillomavirus-positive HeLa cells.Cancer Lett. 2010; 292: 125-132Crossref PubMed Scopus (25) Google Scholar, Little et al., 2013Little A.J. Corbett E. Ortega F. Schatz D.G. Cooperative recruitment of HMGB1 during V(D)J recombination through interactions with RAG1 and DNA.Nucleic Acids Res. 2013; 41: 3289-3301Crossref PubMed Scopus (24) Google Scholar, Polanská et al., 2012Polanská E. Dobšáková Z. Dvořáčková M. Fajkus J. Štros M. HMGB1 gene knockout in mouse embryonic fibroblasts results in reduced telomerase activity and telomere dysfunction.Chromosoma. 2012; 121: 419-431Crossref PubMed Scopus (50) Google Scholar). Critically, HMGBs are depleted from cell nuclei in senescent and aging tissues (Abraham et al., 2013Abraham A.B. Bronstein R. Reddy A.S. Maletic-Savatic M. Aguirre A. Tsirka S.E. Aberrant neural stem cell proliferation and increased adult neurogenesis in mice lacking chromatin protein HMGB2.PLoS ONE. 2013; 8: e84838Crossref PubMed Scopus (23) Google Scholar, Aird et al., 2016Aird K.M. Iwasaki O. Kossenkov A.V. Tanizawa H. Fatkhutdinov N. Bitler B.G. Le L. Alicea G. Yang T.L. Johnson F.B. et al.HMGB2 orchestrates the chromatin landscape of senescence-associated secretory phenotype gene loci.J. Cell Biol. 2016; 215: 325-334Crossref PubMed Scopus (95) Google Scholar, Davalos et al., 2013Davalos A.R. Kawahara M. Malhotra G.K. Schaum N. Huang J. Ved U. Beausejour C.M. Coppe J.P. Rodier F. Campisi J. p53-dependent release of Alarmin HMGB1 is a central mediator of senescent phenotypes.J. Cell Biol. 2013; 201: 613-629Crossref PubMed Scopus (282) Google Scholar, Ly et al., 2000Ly D.H. Lockhart D.J. Lerner R.A. Schultz P.G. Mitotic misregulation and human aging.Science. 2000; 287: 2486-2492Crossref PubMed Scopus (496) Google Scholar, Taniguchi et al., 2009Taniguchi N. Caramés B. Ronfani L. Ulmer U. Komiya S. Bianchi M.E. Lotz M. Aging-related loss of the chromatin protein HMGB2 in articular cartilage is linked to reduced cellularity and osteoarthritis.Proc. Natl. Acad. Sci. USA. 2009; 106: 1181-1186Crossref PubMed Scopus (103) Google Scholar), and markedly overexpressed across cancer types (http://www.cbioportal.org/). Despite their physiological relevance, the binding and roles of HMGBs on chromatin remain enigmatic, but this work sheds new light onto their connection to the proliferative capacity, 3D genome folding, and transcriptional output of primary cells. We hypothesized that different lineages share a common regulatory backbone controlling entry into replicative senescence. To test this, we obtained three distinct primary human cell types from individual donors/isolations: HUVECs (mesodermal) from three single donors, two isolates of IMR90s (endodermal), and MSCs (multipotent) from five donors. As the path to senescence is characterized by heterogeneity (Smith and Whitney, 1980Smith J.R. Whitney R.G. Intraclonal variation in proliferative potential of human diploid fibroblasts: stochastic mechanism for cellular aging.Science. 1980; 207: 82-84Crossref PubMed Scopus (233) Google Scholar), we used phenotypic and molecular markers to define the passage at which ∼65% of cells in each population had entered senescence. Cells staining positive for β-galactosidase, showing significantly reduced proliferation, and specific methylation changes at six senescence-predictive CpGs (Franzen et al., 2017Franzen J. Zirkel A. Blake J. Rath B. Benes V. Papantonis A. Wagner W. Senescence-associated DNA methylation is stochastically acquired in subpopulations of mesenchymal stem cells.Aging Cell. 2017; 16: 183-191Crossref PubMed Scopus (49) Google Scholar) were deemed senescent (Figures 1A, S1A, and S1B). Note here that one HUVEC and one MSC donor displayed a limited number of population doublings until senescence and, despite their convergent gene expression profiles, were excluded from further analyses. This highlights the idiosyncratic nature of cellular aging due to donor-specific features. Total RNA from proliferating and senescent populations from all cell types and donors was collected, depleted of rRNA, poly(A)+-enriched (except for HUVECs), and sequenced to >50 million read pairs each. Following mapping to the genome (hg19), read counts were normalized in silico to control for differences in transcript abundance as a result of senescence entry (Risso et al., 2014Risso D. Ngai J. Speed T.P. Dudoit S. Normalization of RNA-seq data using factor analysis of control genes or samples.Nat. Biotechnol. 2014; 32: 896-902Crossref PubMed Scopus (893) Google Scholar; Figure S1C). Differential gene expression analysis revealed different numbers of up- and downregulated genes in each cell type (±0.6 log2 fold change), which nonetheless showed converging gene ontology (GO) profiles (Figures S1D and S1E). A total of 153 upregulated genes, mostly involved in cell growth, p53 responses, and ECM reorganization, were shared by all cell types (Figure S1F). On the other hand, the 206 shared downregulated genes were associated with cell-cycle regulation, replication, and DNA metabolism, but critically also with DNA conformation, chromatin organization, and DNA packaging (Figure 1B). The latter terms are due to genes like HMGB1/B2, TOP2A, NCAPD/-G/-H, or ASF1, and aligned with our hypothesis on 3D chromatin reorganization upon senescence entry. We validated gene expression changes by comparison to available data from senescent s (Rai et al., 2014Rai T.S. Cole J.J. Nelson D.M. Dikovskaya D. Faller W.J. Vizioli M.G. Hewitt R.N. Anannya O. McBryan T. Manoharan I. et al.HIRA orchestrates a dynamic chromatin landscape in senescence and is required for suppression of neoplasia.Genes Dev. 2014; 28: 2712-2725Crossref PubMed Scopus (90) Google Scholar; Figure S1G), and by time course qRT-PCR on selected targets (Figure S1H). We also isolated and analyzed nascent RNA from proliferative/senescent IMR90s via “factory” RNA sequencing (RNA-seq) (Melnik et al., 2016Melnik S. Caudron-Herger M. Brant L. Carr I.M. Rippe K. Cook P.R. Papantonis A. Isolation of the protein and RNA content of active sites of transcription from mammalian cells.Nat. Protoc. 2016; 11: 553-565Crossref PubMed Scopus (8) Google Scholar) to show that most regulation occurs at the level of transcription (Figures 1C and S1I). Due to their ability to bend DNA in vitro, their reduced expression in senescent and aging tissue in vivo, and their unknown roles on chromatin, we focused on HMGB1/B2 and verified their suppression at the protein level in both IMR90s and HUVECs (Figure 1D). Transcriptional heterogeneity is inherent to senescent cell populations (Hernandez-Segura et al., 2017Hernandez-Segura A. de Jong T.V. Melov S. Guryev V. Campisi J. Demaria M. Unmasking transcriptional heterogeneity in senescent cells.Curr. Biol. 2017; 27: 2652-2660.e4Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). To show that HMGB1/B2 are suppressed before known senescence markers appear, we performed single-cell mRNA sequencing (scRNA-seq) by analyzing ∼8,300 proliferating and ∼5,200 senescent HUVECs on a 10X Genomics platform. Following multiplexed sequencing, we generated ∼30,000 reads per cell in either state, and >2,500 individual transcripts were robustly captured per cell. Next, we clustered cells from each state based on their individual expression profiles. Unsupervised clustering showed proliferating and senescent cells segregating into four clusters (Figure 2A). Interestingly, the three major senescent cell clusters reflected the relative distributions of HMGB2 and CDKN1A (p21) expression: low HMGB2 levels mostly marked cells with medium-to-high CDKN1A levels (i.e., replicatively arrested cells), yet >1,800 cells carried little HMGB2 mRNA without CDKN1A induction. Similarly, >2,500 proliferating cells showed robust HMGB1 but already low HMGB2 levels (Figures 2B and 2C). Senescent cells lacking HMGB2 expression were also marked by high levels of the IL6 and IL8 SASP genes (Figure 2B). Importantly, the gene expression profiles previously reported to hold key roles in senescence and the cell cycle, like LMNB1, EZH2, PCNA, or CCNA2, also differentially marked proliferating and senescent subpopulations, but were less informative for clustering (Figure 2B). Last, we exploited the heterogeneity in HUVEC populations to perform pseudo-time course analyses of scRNA-seq data (Haghverdi et al., 2016Haghverdi L. Büttner M. Wolf F.A. Buettner F. Theis F.J. Diffusion pseudotime robustly reconstructs lineage branching.Nat. Methods. 2016; 13: 845-848Crossref PubMed Scopus (508) Google Scholar). We found that the gradual drop in HMGB1/B2 levels in proliferating HUVECs sufficed for describing seven states that appear consecutively derived from one another (Figure 2D). On the other hand, the eight senescent cell states along the main path display variable amounts of HMGB1/B2 and are best described by a rise in CDKN1A levels and by strong IL8 and CXCL1 SASP gene expression. Still, subclusters 1 and 4 had HMGB2 suppressed, but HMGB1 robustly detected (Figure 2D). This, to our knowledge, first ever transcriptome of single replicatively senescent cells documents how HMGB2 suppression is an early event marking senescence entry. To complement scRNA-seq data and examine changes in HMGB1/B2 titers, we turned to single-cell immunofluorescence. Super-resolution imaging found the abundant HMGB1/B2 proteins localizing in non-DAPI-dense nuclear areas (Figure S2A). Widefield microscopy coupled to semi-automated quantification of fluorescence levels (STAR Methods) revealed that senescent nuclei almost doubled in area across cell types, and larger nuclei were almost invariably β-galactosidase positive (Figures 3A and 3B ). This urged us to stratify all microscopy data according to increasing nuclear sizes. This way we could verify that HMGB1/B2 were depleted from essentially all large HUVEC, IMR90, or MSC nuclei (Figure 3C). Although HMGB1/B2 nuclear depletion appeared coordinated among individual cells (ρ = 0.81), p21 upregulation—a hallmark of replicative arrest—correlated rather poorly with low HMGB2 titers (ρ = 0.35; Figures 3D and 3E). This agrees with our scRNA-seq data in which HUVECs with low HMGB2 titers and no CDKN1A and/or robust HMGB1 levels represent an early state on the path to senescence. Focusing on size-discretized nuclei, we found that larger and HMGB2-depleted nuclei show specific decrease in H3K27me3 levels, the mark characteristic of facultative heterochromatin (Shah et al., 2013Shah P.P. Donahue G. Otte G.L. Capell B.C. Nelson D.M. Cao K. Aggarwala V. Cruickshanks H.A. Rai T.S. McBryan T. et al.Lamin B1 depletion in senescent cells triggers large-scale changes in gene expression and the chromatin landscape.Genes Dev. 2013; 27: 1787-1799Crossref PubMed Scopus (353) Google Scholar), while also displaying increased HP1α levels (marking constitutive heterochromatin, but without SAHF formation as in OIS; Narita et al., 2006Narita M. Narita M. Krizhanovsky V. Nuñez S. Chicas A. Hearn S.A. Myers M.P. Lowe S.W. A novel role for high-mobility group a proteins in cellular senescence and heterochromatin formation.Cell. 2006; 126: 503-514Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar; Figure S2B). This was concomitant with a drop in histone acetylation measured using H3K27ac or H4K16ac as markers (Figures S2C and S2D). Finally, increased H3K9 methylation was confirmed in senescent HUVECs and IMR90s via ELISA (Figure S2E), while the senescent-induced drop in H3K27ac/me3 was in line with changes in H3K27ac/me3 chromatin immunoprecipitation sequencing (ChIP-seq) signal (Figures S2F and S2G). Population and single-cell data encouraged us to interrogate changes in whole-genome 3D folding upon senescence entry via Hi-C. We used HindIII and obtained >300 million read pairs per donor and condition, after sequencing two technical replicates from two HUVEC and IMR90 donors/isolates and one MSC donor (Figure S3A). After confirming reproducibility between Hi-C maps from the same cell type and condition (Figure S3B), we merged replicates and could now afford ∼20-kbp resolution of spatial chromatin interactions in both proliferating and senescent cells (higher than that afforded for “deep” senescence; Criscione et al., 2016Criscione S.W. De Cecco M. Siranosian B. Zhang Y. Kreiling J.A. Sedivy J.M. Neretti N. Reorganization of chromosome architecture in replicative cellular senescence.Sci. Adv. 2016; 2: e1500882Crossref PubMed Scopus (81) Google Scholar). Initially, we looked into interchromosomal interaction changes between whole chromosomes upon senescence entry; both HUVEC and IMR90 chromosomes mostly displayed decreased, cell-type-specific trans-interactions (Figure S3C). We next examined intrachromosomal interactions. Two features stand out when comparing Hi-C interaction maps from proliferating and senescent HUVECs: first, stronger longer-range interactions emerge upon senescence entry; second, apparent insulation, especially between large higher-order domains, is seen in senescent cells (Figures 4A and S4A). This holds true across all cell types and chromosomes studied (Figures S3D and S4A–S4C). Interaction changes between single donors/isolates visualized at a 200 kbp resolution are almost invariably of the same nature (Figure S3E). But when different cell types are compared, the same chromosomal regions refold in a cell-type-specific manner upon senescence (Figures 4B and S4F), and this is in line with senescence epigenetically reprogramming each cell type. In addition, most Hi-C data to date display more changes at the level of A/B compartments than at that of TADs (e.g., Criscione et al., 2016Criscione S.W. De Cecco M. Siranosian B. Zhang Y. Kreiling J.A. Sedivy J.M. Neretti N. Reorganization of chromosome architecture in replicative cellular senescence.Sci. Adv. 2016; 2: e1500882Crossref PubMed Scopus (81) Google Scholar, Dixon et al., 2012Dixon J.R. Selvaraj S. Yue F. Kim A. Li Y. Shen Y. Hu M. Liu J.S. Ren B. Topological domains in mammalian genomes identified by analysis of chromatin interactions.Nature. 2012; 485: 376-380Crossref PubMed Scopus (3981) Google Scholar). However, we observed limited switching between A/B compartments (Figures 4B and S5A). We went on to define TADs in our Hi-C data from both conditions across cell types. We employed “TADtool” (Kruse et al., 2016Kruse K. Hug C.B. Hernández-Rodríguez B. Vaquerizas J.M. TADtool: visual parameter identification for TAD-calling algorithms.Bioinformatics. 2016; 32: 3190-3192Crossref PubMed Scopus (37) Google Scholar) and the resolution afforded by our lowest-covered dataset (40-kbp resolution in MSCs), while applying similar TAD-calling parameters for proliferating or senescent data in each cell type. This analysis returned a total of ∼3,000 TADs for HUVECs and IMR90s and ∼3,500 for MSCs in either state. In IMR90s and MSCs, ∼50% of TADs remain unchanged upon senescence entry, while ∼20% shift at least one of their boundaries by ≥80 kbp and ∼25% fuse into larger TADs (Figure 4C). TAD changes were more drastic in HUVECs, with 24% TADs shifting boundaries and 43% fusing into larger ones—in agreement with the strong differences seen in HUVEC Hi-C (Figures S4A–S4C). Interaction decay plots revealed widespread changes in both longer- and shorter-range interactions upon senescence entry (Figures 4D and S5B), which differs from what was observed for “deep” senescence (Criscione et al., 2016Criscione S.W. De Cecco M. Siranosian B. Zhang Y. Kreiling J.A. Sedivy J.M. Neretti N. Reorganization of chromosome architecture in replicative cellular senescence.Sci. Adv. 2016; 2: e1500882Crossref PubMed Scopus (81) Google Scholar; Figures S5C and S5D). Finally, we used 3D DNA-fluorescence in situ hybridization (FISH) (Roukos et al., 2015Roukos V. Pegoraro G. Voss T.C. Misteli T. Cell cycle staging of individual cells by fluorescence microscopy.Nat. Protoc. 2015; 10: 334-348Crossref PubMed Scopus (99) Google Scholar), with probes targeting three increasingly separated regions on the long arm of chr12 to query conformational changes. In senescent HUVECs, which are predominantly tetraploid (Figure S6A), FISH showed all probes becoming more separated from one another upon senescence and assuming less peripheral positioning in the larger senescent nuclei (Figures S6B–S6E). In IMR90s, which remain diploid, the two most distant probes come significantly closer together upon senescence, while the intervening one separates out, and only one probe pair shows less peripheral positioning (Figures S9A–S9E). Critically, in both HUVECs and IMR90s the FISH data agree with changes seen by Hi-C (i.e., increased probe separation matched by decreased interaction frequency; Figures S6C and S6E). Genome reorganization in OIS and in “deep” senescence involves formation of characteristic SAHFs (Chandra et al., 2015Chandra T. Ewels P.A. Schoenfelder S. Furlan-Magaril M. Wingett S.W. Kirschner K. Thuret J.Y. Andrews S. Fraser P. Reik W. Global reorganization of the nuclear landscape in senescent cells.Cell Rep. 2015; 10: 471-483Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar) and strong chromatin compaction, respectively (Criscione et al., 2016Criscione S.W. De Cecco M. Siranosian B. Zhang Y. Kreiling J.A. Sedivy J.M. Neretti N. Reorganization of chromosome architecture in replicative cellular senescence.Sci. Adv. 2016; 2: e1500882Crossref PubMed Scopus (81) Google Scholar). Here, senescence entry coincides with a heterochromatic shift (Figure S2B), and we asked whether this suffices to explain the changes seen by Hi-C (Figure S4). As it is not straightforward to experimentally decouple HMGB1/B2 loss from heterochromatic changes, we used molecul" @default.
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• W2799540870 date "2018-05-01" @default.
• W2799540870 modified "2023-10-14" @default.
• W2799540870 title "HMGB2 Loss upon Senescence Entry Disrupts Genomic Organization and Induces CTCF Clustering across Cell Types" @default.
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• W2799540870 doi "https://doi.org/10.1016/j.molcel.2018.03.030" @default.
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• W2799540870 hasPublicationYear "2018" @default.

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