Nuclear expansion drives chromatin structure remodeling in aging neurons

Nuclear expansion drives chromatin structure remodeling in aging neurons | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Nuclear expansion drives chromatin structure remodeling in aging neurons Debra Toiber, Dmitrii Kruikov, Ekaterina Eremenko, Dmitrii Smirnov, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3691075/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Aging, particularly in the brain, involves impairments in multiple cellular and molecular functions, many of which are regulated at the nucleus. Chromatin structure plays a critical role in the regulation of gene expression and the maintenance of genomic stability. During differentiation, chromosomes acquire their unique topology depending on the cell type that should be kept for a lifetime, but this may deteriorate as we age. However, the effects of aging on the chromatin 3D structure of neurons remain largely unknown and much has been inferred from senescent cells. By combining chromosome conformation capture and microscopy techniques, we investigated cortical neurons of young and aged mice and discovered neuronal nuclear expansion during neuronal aging, leading to increased distances between chromosomes. This expansion alters the topology of compartments, topologically associating domains (TADs) and chromatin loops. While larger TADs tend to dissociate, smaller TADs and loops exhibit strengthened interactions to maintain the cohesiveness of chromatin in aged neurons. These topological changes impact the borders of TADs, resulting in their overall weakening. Interestingly, we attribute these alterations to changes in the physical forces of an expanding nucleus, filling a growing nuclear area, affecting downstream gene expression and chromatin topology, further contributing to the functional declines observed during aging. Biological sciences/Molecular biology/Chromatin/Chromatin structure Biological sciences/Neuroscience/Cellular neuroscience Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Full Text Additional Declarations There is NO Competing Interest. Supplementary Files Supplementaryfigures.pdf abstract.png Graphical Abstract During aging, cortical neurons nuclei lose lamin-B, deforming, losing eccentricity, and expanding, resulting in chromosomes distancing from each other. The large TADs are broadening, the expansion is more prominent near the nuclear membrane, where the distancing of chromosomes is higher compared to the nucleus center, and the extension of B domains into the Lamin-B associated regions across larger areas. While highly expressed genes in small TADs become tighter, keeping their structure and borders, loss in the larger regions. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3691075","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":256237335,"identity":"4cb13151-5674-4c29-931b-3a6358121d57","order_by":0,"name":"Debra Toiber","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuUlEQVRIiWNgGAWjYFACxjYgYZMA5UnIENTAA9GSBtfCQ4QWBjYgdTgBSYAAsGdvbnvwcc/5PPmIBMYPPxgsiLCF52C74Yxnt4sNbyQwS/YQ5TCJxDZpngO3EzfOSGCQJs4vIC1/DpwDaWH+TbwWhgMHEudLJLARacsZoF96DiQXG/A8bLPsMSBCC3t7+7MHPw7Y5cm3Jx++8aOiTo6gFjgwOMDYACSJ18DAIN9AiupRMApGwSgYUQAAZRY2eAVtP24AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-1465-0130","institution":"Ben Gurion University of the Negev","correspondingAuthor":true,"prefix":"","firstName":"Debra","middleName":"","lastName":"Toiber","suffix":""},{"id":256237336,"identity":"8a6fd16e-282b-40a1-8c9f-94e16c9d3ae8","order_by":1,"name":"Dmitrii Kruikov","email":"","orcid":"","institution":"Skolkovo Institute of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Dmitrii","middleName":"","lastName":"Kruikov","suffix":""},{"id":256237337,"identity":"b5c08899-e0c8-4a6d-a000-294df4586a1e","order_by":2,"name":"Ekaterina Eremenko","email":"","orcid":"","institution":"Department of Life Sciences, Ben-Gurion University of the Negev","correspondingAuthor":false,"prefix":"","firstName":"Ekaterina","middleName":"","lastName":"Eremenko","suffix":""},{"id":256237338,"identity":"55eb7e02-1923-47db-b2ce-de45cce981d5","order_by":3,"name":"Dmitrii Smirnov","email":"","orcid":"","institution":"Department of Life Sciences, Ben-Gurion University of","correspondingAuthor":false,"prefix":"","firstName":"Dmitrii","middleName":"","lastName":"Smirnov","suffix":""},{"id":256237339,"identity":"6a74841c-b91b-49e5-a504-e5eb8b5f9a3f","order_by":4,"name":"Daniel Stein","email":"","orcid":"","institution":"Department of Life Sciences, Ben-Gurion University of","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"","lastName":"Stein","suffix":""},{"id":256237340,"identity":"bd894351-13a5-489f-914e-119c23c58e2a","order_by":5,"name":"Alexandra Tsitrina","email":"","orcid":"","institution":"Department of Life Sciences, Ben-Gurion University of","correspondingAuthor":false,"prefix":"","firstName":"Alexandra","middleName":"","lastName":"Tsitrina","suffix":""},{"id":256237341,"identity":"91f9ab3f-d726-41f2-ae10-32fe4a005246","order_by":6,"name":"Anastasia Golova","email":"","orcid":"","institution":"Center for Molecular and Cellular Biology, Skolkovo Institute","correspondingAuthor":false,"prefix":"","firstName":"Anastasia","middleName":"","lastName":"Golova","suffix":""},{"id":256237342,"identity":"3fa2b9c4-5cdf-4e33-847a-70fd70633a9b","order_by":7,"name":"Monica Einav","email":"","orcid":"","institution":"Department of Life Sciences, Ben-Gurion University of the Negev","correspondingAuthor":false,"prefix":"","firstName":"Monica","middleName":"","lastName":"Einav","suffix":""},{"id":256237343,"identity":"235ba38f-7e6a-4cc3-98e4-5f476816634b","order_by":8,"name":"Ekaterina Khrameeva","email":"","orcid":"","institution":"Center for Molecular and Cellular Biology, Skolkovo Insti","correspondingAuthor":false,"prefix":"","firstName":"Ekaterina","middleName":"","lastName":"Khrameeva","suffix":""}],"badges":[],"createdAt":"2023-12-01 08:10:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3691075/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3691075/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":48075997,"identity":"a80b841e-ae80-49e6-84a3-788ca67eab30","added_by":"auto","created_at":"2023-12-12 16:00:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1917202,"visible":true,"origin":"","legend":"\u003cp\u003eGenome-scale changes of chromatin folding in the aging mouse neurons. (A) The experimental procedure implemented in this study. Neurons were isolated using FACS from the mouse cortex tissue. (B) The design of this study. Hi-C experiments were performed in neurons of young (4 months), adult (10 months), and old (20 months) mice in three replicates per age group. (C) Levels of chromatin architecture in our data that are being studied for age-related changes: inter- and intra-chromosomal contacts, A/B compartments, TADs and loops. (D) Age-related changes of interactions within all chromosomes (cis contacts, orange boxplots) and between all pairs of chromosomes (trans contacts, blue boxplots) calculated as the contact ratio between old and adult mice, adult and young mice. Asterisks indicate Wilcoxon test p-values: **** - p \u0026lt; 0.00001, *** - p \u0026lt; 0.0001, ns - p \u0026gt; 0.05. (E) Hierarchical clustering of chromosomes by their contact ratio Old/Adult. NOR chromosomes are highlighted with the red rectangle. (F) The schematic illustrating genome-scale changes of chromatin folding in the aging neurons: inter-chromosomal contacts strongly decrease, while intra-chromosomal contacts slightly increase in the expanded nucleus volume.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3691075/v1/a46705666fe6e7cfb8756230.png"},{"id":48075994,"identity":"650bebd3-edfe-4c83-96bf-a1334c4dcbcb","added_by":"auto","created_at":"2023-12-12 16:00:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":572324,"visible":true,"origin":"","legend":"\u003cp\u003eChromatin architecture is altered at the chromosomal scale during aging. (A) Clustering analysis of age groups based on the first eigenvectors representing compartments. (B) Differences in inter-compartment (AB) and intra-compartment (AA, BB) interactions, calculated between old and adult mice. Colors represent the fold change of interactions between the age groups. (C) Differences in interactions between old and adult, adult and young mice within areas highlighted in panel B. The dashed line corresponds to the absence of changes. (D) The schematic summarizing aging-related compartment changes in our data. (E) Percentage of A (red) and B (blue) compartments in LADs (left panel) and outside LADs (right panel. (F) The schematic illustrating the expansion of B compartment and reduction of A compartment upon aging. (G) The ratio of intrachromosomal interactions calculated between adult and young (blue), old and adult (orange) mice highlighting aging-related polymer scaling changes. The dashed line corresponds to the absence of changes. The schematic on top illustrates typical chromatin organization features corresponding to the genomic distances.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-3691075/v1/3721a14c0bfde344fa9a9df5.png"},{"id":48076001,"identity":"a3d6dad2-b6e2-4524-aab4-3ee8f8cbd80e","added_by":"auto","created_at":"2023-12-12 16:00:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3586460,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological and Lamin B Aberrations in Aged Neuronal Nuclei A. Schematic representation illustrating the isolation of neuronal nuclei from young (n=6), adult (n=5), and old (n=4) mice brains. Neurons were extracted, stained with NeuN-PE and anti-Lamin B antibodies for subsequent analysis. B. Confocal Imaging of representative nuclei. C. Quantification of Nuclear area D. Quantification of Nuclear Eccentricity E. Representative confocal images for lamin B intensity (upper panel) and lamin B abnormalities (lower panel) F. Lamin B intensity (MFI, mean fluorescence intensity) (ns: 0.05\u0026lt;𝑝 ≤1.0, *: 0.01\u0026lt;𝑝 ≤0.05, **: 0.001\u0026lt;𝑝 ≤0.01, ***: 0.0001\u0026lt;𝑝 ≤0.001, ****: 𝑝≤0.0001). G. Quantification of Lamin B abnormalities in individual nuclei from different age groups (young, adults, and old) using a 5-grade scoring system. Aberrations were evaluated based on circularity, blebbing, mislocalization, presence of foci, and envelope integrity (𝜒2(𝑑𝑓=10)=142.77,𝑝−𝑣𝑎𝑙𝑢𝑒\u0026lt;2.2×10−16).\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-3691075/v1/7dd7fa19294440ed2b166e05.png"},{"id":48076000,"identity":"3e34a551-6000-494e-b65a-b5171da93453","added_by":"auto","created_at":"2023-12-12 16:00:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1939425,"visible":true,"origin":"","legend":"\u003cp\u003eSmall TADs and loops have more interactions in aging neurons. (A) Clustering analysis based on the number of common TAD boundaries between the age groups. (B) Venn diagram of TAD boundaries shared between the age groups showing that the old mice are the most divergent. (C) Average interaction differences between old and adult mice (left panel), adult and young mice (right panel) at all TAD boundaries. (D) Average interaction differences between old and adult mice calculated separately for small, medium and large TADs. (E) Boxplots of contact ratio Old/Adult calculated for small, medium and large TADs. Stars indicate significant difference from ratio=1 (**** - P-value \u0026lt; 0.0001, Wilcoxon one-sample test). (F,K) Gene expression in TADs (F) or loop anchors (K) of different sizes. Lines represent average values, shaded areas - 95% confidence intervals for the average estimates. (G,L) Number of HK genes in TADs (G) or loop anchors (L) of different sizes. (H) Average interaction differences between old and adult mice calculated separately for TADs overlapping (left panels) and not overlapping (right panels) LADs. (I-J) Average interaction differences between old and adult mice at loop positions: for all loops combined (I) and for short-, mid- and long-range loops separately (J).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-3691075/v1/a843b3ce3a26a9d32dff4dbe.png"},{"id":48075996,"identity":"75a56ff7-8903-4947-b4e7-26cf9ac4e6c2","added_by":"auto","created_at":"2023-12-12 16:00:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":507438,"visible":true,"origin":"","legend":"\u003cp\u003eATAC-seq reveals global alterations in regulatory landscape during aging.\u003c/p\u003e\n\u003cp\u003e(A) ATAC-seq experiments were performed in neurons of the same mice: young (4 months, n=3), adult (10 months, n=3), and old (20 months, n=3). (B) The distribution of peak positions across the mouse genome for peaks emerging in old mice (N=4460) and peaks vanishing in old mice (N=8441). Purple bars show percents of peaks within a particular annotation group, while grey bars show the percentage expected by chance. Error bars represent 95% confidence intervals on the average peak count. Percentages near bars represent increase or decrease compared to expected values. Asterisks indicate whether the change is significant (ns: 0.05\u0026lt;𝑝 ≤1.0, *: 0.01\u0026lt;𝑝 ≤0.05, **: 0.001\u0026lt;𝑝 ≤0.01, ***: 0.0001\u0026lt;𝑝 ≤0.001, ****: 𝑝≤0.0001). (C) Functional analysis of peaks emerging in old mice based on the KEGG annotation. Each peak is assigned to the nearest gene. The size of circles represents the number of genes in the KEGG pathway, while the color shows p-values. (D-F) The percentage of all promoter peaks (D), emerging non-promoter peaks (E), vanishing non-promoter peaks (F) in old mice within anchors of loops of different sizes defined in Fig. 4J.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-3691075/v1/9d3e4696e3b938b0dc2ef043.png"},{"id":48075992,"identity":"0782fc30-8d5a-43bb-92da-486650d79a6e","added_by":"auto","created_at":"2023-12-12 16:00:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":279808,"visible":true,"origin":"","legend":"\u003cp\u003eScheme of dedifferentiation hypothesis where cells are acquiring an adult differentiated topology, which is reversed during aging, becoming similar to the young immature neurons.\u003c/p\u003e","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-3691075/v1/a87344e06c8ba21ffdc7fe5d.png"},{"id":50786856,"identity":"ab4f43b7-281e-485c-8748-6870248d98b4","added_by":"auto","created_at":"2024-02-07 09:40:15","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1027510,"visible":true,"origin":"","legend":"","description":"","filename":"KriukovEremenkoFINAL.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3691075/v1_covered_2af36735-e3bc-4fae-8150-2bc3ce8ad6e7.pdf"},{"id":48076007,"identity":"62e43e60-23c5-474f-9a15-43d0c484e21a","added_by":"auto","created_at":"2023-12-12 16:00:46","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1245592,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Supplementaryfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3691075/v1/17d27d4b98d3cace7b91670d.pdf"},{"id":48075995,"identity":"9f82b3e3-6ba8-4072-a664-15b6d375276a","added_by":"auto","created_at":"2023-12-12 16:00:36","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1043032,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical Abstract During aging, cortical neurons nuclei lose lamin-B, deforming, losing eccentricity, and expanding, resulting in chromosomes distancing from each other. The large TADs are broadening, the expansion is more prominent near the nuclear membrane, where the distancing of chromosomes is higher compared to the nucleus center, and the extension of B domains into the Lamin-B associated regions across larger areas. While highly expressed genes in small TADs become tighter, keeping their structure and borders, loss in the larger regions.\u003c/p\u003e","description":"","filename":"abstract.png","url":"https://assets-eu.researchsquare.com/files/rs-3691075/v1/bf409bb34125b31302216854.png"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Nuclear expansion drives chromatin structure remodeling in aging neurons","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3691075/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3691075/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Aging, particularly in the brain, involves impairments in multiple cellular and molecular functions, many of which are regulated at the nucleus. Chromatin structure plays a critical role in the regulation of gene expression and the maintenance of genomic stability. During differentiation, chromosomes acquire their unique topology depending on the cell type that should be kept for a lifetime, but this may deteriorate as we age. However, the effects of aging on the chromatin 3D structure of neurons remain largely unknown and much has been inferred from senescent cells. By combining chromosome conformation capture and microscopy techniques, we investigated cortical neurons of young and aged mice and discovered neuronal nuclear expansion during neuronal aging, leading to increased distances between chromosomes. This expansion alters the topology of compartments, topologically associating domains (TADs) and chromatin loops. While larger TADs tend to dissociate, smaller TADs and loops exhibit strengthened interactions to maintain the cohesiveness of chromatin in aged neurons. These topological changes impact the borders of TADs, resulting in their overall weakening. Interestingly, we attribute these alterations to changes in the physical forces of an expanding nucleus, filling a growing nuclear area, affecting downstream gene expression and chromatin topology, further contributing to the functional declines observed during aging.","manuscriptTitle":"Nuclear expansion drives chromatin structure remodeling in aging neurons","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-12-12 15:59:41","doi":"10.21203/rs.3.rs-3691075/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b92046ce-de34-4b64-b20f-5e73e1cb599d","owner":[],"postedDate":"December 12th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":27063668,"name":"Biological sciences/Molecular biology/Chromatin/Chromatin structure"},{"id":27063669,"name":"Biological sciences/Neuroscience/Cellular neuroscience"}],"tags":[],"updatedAt":"2024-02-07T09:31:59+00:00","versionOfRecord":[],"versionCreatedAt":"2023-12-12 15:59:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3691075","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3691075","identity":"rs-3691075","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}