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Cited by 29Genomics and Chromatin DynamicsChromatin Remodeling and CancerCancer-related molecular mechanisms research

An interdependent network of functional enhancers regulates transcription and EZH2 loading at the INK4a/ARF locus

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Farooq, Umer, Saravanan, Bharath, Islam, Zubairul, Walavalkar, Kaivalya, Singh, Anurag Kumar, Jayani, Ranveer Singh, Meel, Sweety, Swaminathan, Sudha +1 more·2021·Cell Reports
DOI 10.1016/j.celrep.2021.108898PMC PMC7611173
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Abstract

. The neighboring gene desert to this locus is the most reproducible GWAS hotspot that harbors one of the densest enhancer clusters in the genome. However, how multiple enhancers that overlap with GWAS variants regulate the INK4a/ARF locus is unknown, which is an important step in linking genetic variation with associated diseases. Here, we show that INK4a/ARF promoters interact with a subset of enhancers in the cluster, independent of their H3K27ac and eRNA levels. Interacting enhancers transcriptionally control each other and INK4a/ARF promoters over long distances as an interdependent single unit. The deletion of even a single interacting enhancer results in an unexpected collapse of the entire enhancer cluster and leads to EZH2 enrichment on promoters in an ANRIL-independent manner. Dysregulated genes genome-wide mimic 9p21-associated diseases under these scenarios. Our results highlight intricate dependencies of promoter-interacting enhancers on each other.

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  • In the INK4a/ARF locus, perturbing any promoter-interacting enhancer within the chain abolished functionality of the entire network, but deleting enhancers further down the chain had more moderate effects.

    “recent dissection of an enhancer chain in the INK4a/ARF locus revealed that perturbing any promoter-interacting enhancer within the chain abolished the functionality of the entire network, but deleting the enhancers further down the chain had more moderate effects [491].”

    From Transcriptional enhancers and their communication with gene promoters (2021) · cites this paper as [491]

    The Results (‘An interdependent enhancer network operates within the enhancer cluster’; page unavailable) report that blocking any of the three tested promoter-interacting enhancers reduced activity of the others. The Results (‘Each promoter-interacting enhancer fully regulates INK4a/ARF genes’; page unavailable) report that deleting E21 had only marginal effects on ARF and INK4b and no effect on INK4a. But E21 is described as a non-promoter-interacting enhancer, not as one of multiple enhancers ‘further down the chain’; the paper describes the interacting network as equal-weighted rather than a directional chain. (LLM, conf=0.87)

    Experimental — accuracy not yet measured

  • Deletion or inhibition of any one of the promoter-interacting enhancers caused disruption of H3K27ac and eRNA levels on the other enhancers in the network.

    “deletion or inhibition of any one of the promoter-interacting enhancers caused disruption of H3K27ac and eRNA levels on the other enhancers in the network [491].”

    From Transcriptional enhancers and their communication with gene promoters (2021) · cites this paper as [491]

  • In some cases, several enhancers form a functionally interdependent network, so that exclusion of any single enhancer from this network results in loss of all enhancer activity.

    “In some cases, several enhancers form a functionally interdependent network, so that the exclusion of any single enhancer from this network results in the loss of all enhancer activity [27].”

    From Enhancer Function in the 3D Genome (2023) · cites this paper as [27]

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Research works printed but not linked: 61

  1. Aguilo F Zhou MM Walsh MJ Long noncoding RNA, polycomb, and the ghosts haunting INK4b-ARF-INK4a expression Cancer Res 2011 71 5365 5369 21828241 PMC3339196
  2. Aloia L Demajo S Di Croce L ZRF1: a novel epigenetic regulator of stem cell identity and cancer Cell Cycle 2015 14 510 515 25665097 PMC4615000
  3. Beltran M Yates CM Skalska L Dawson M Reis FP Viiri K Fisher CL Sibley CR Foster BM Bartke T The interaction of PRC2 with RNA or chromatin is mutually antagonistic Genome Res 2016 26 896 907 27197219 PMC4937559
  4. Carrasco-Garcia E Arrizabalaga O Serrano M Lovell-Badge R Matheu A Increased gene dosage of Ink4/Arf and p53 delays age-associated central nervous system functional decline Aging Cell 2015 14 710 714 25990896 PMC4531087
  5. Cifuentes-Rojas C Hernandez AJ Sarma K Lee JT Regulatory interactions between RNA and polycomb repressive complex 2 Mol Cell 2014 55 171 185 24882207 PMC4107928
  6. Corces MR Granja JM Shams S Louie BH Seoane JA Zhou W Silva TC Groeneveld C Wong CK Cho SW The chromatin accessibility landscape of primary human cancers Science 2018 80 362 PMC6408149 30361341
  7. Creyghton MP Cheng AW Welstead GG Kooistra T Carey BW Steine EJ Hanna J Lodato MA Frampton GM Sharp PA Histone H3K27ac separates active from poised enhancers and predicts developmental state Proc Natl Acad Sci USA 2010 107 21931 21936 21106759 PMC3003124
  8. Deloukas P Kanoni S Willenborg C Farrall M Assimes TL Thompson JR Ingelsson E Saleheen D Erdmann J Goldstein BA CARDIo-GRAMplusC4D Consortium; DIAGRAM Consortium; CARDIOGENICS Consortium; MuTHER Consortium; Wellcome Trust Case Control Consortium Large-scale association analysis identifies new risk loci for coronary artery disease Nat Genet 2013 45 25 33 23202125 PMC3679547
  9. Drak Alsibai K Vacher S Meseure D Nicolas A Lae M Schnitzler A Chemlali W Cros J Longchampt E Cacheux W High positive correlations between ANRIL and p16-CDKN2A/p15-CDKN2B/p14-ARF gene cluster overexpression in multi-tumor types suggest deregulated activation of an ANRIL-ARF bidirectional promoter Noncoding RNA 2019 5 44 PMC6789474 31438464
  10. Gamell C Ginsberg D Haupt S Haupt Y New insights on the regulation of INK4/ARF locus expression Oncotarget 2017 8 106147 106148 29290925 PMC5739710
  11. Gilbert LA Horlbeck MA Adamson B Villalta JE Chen Y Whitehead EH Guimaraes C Panning B Ploegh HL Bassik MC Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation Cell 2014 159 647 661 25307932 PMC4253859
  12. Gonzalez S Serrano M Anew mechanism of inactivation of the INK4/ARF locus Cell Cycle 2006 5 1382 1384 16855387
  13. González-Navarro H Vinué Á Sanz MJ Delgado M Pozo MA Serrano M Burks DJ Andrés V Increased dosage of Ink4/ Arf protects against glucose intolerance and insulin resistance associated with aging Aging Cell 2013 12 102 111 23107464
  14. Harismendy O Notani D Song X Rahim NG Tanasa B Heintzman N Ren B Fu XD Topol EJ Rosenfeld MG Frazer KA 9p21 DNA variants associated with coronary artery disease impair interferon-g signalling response Nature 2011 470 264 268 21307941 PMC3079517
  15. Helgadottir A Thorleifsson G Magnusson KP Grétarsdottir S Steinthorsdottir V Manolescu A Jones GT Rinkel GJE Blankensteijn JD Ronkainen A The same sequence variant on 9p21 associates with myocardial infarction, abdominal aortic aneurysm and intracranial aneurysm Nat Genet 2008 40 217 224 18176561
  16. Hirosue A Ishihara K Tokunaga K Watanabe T Saitoh N Nakamoto M Chandra T Narita M Shinohara M Nakao M Quantitative assessment of higher-order chromatin structure of the INK4/ARF locus in human senescent cells Aging Cell 2012 11 553 556 22340434
  17. Hnisz D Abraham BJ Lee TI Lau A Saint-André V Sigova AA Hoke HA Young RA Super-enhancers in the control of cell identity and disease Cell 2013 155 934 947 24119843 PMC3841062
  18. Hosogane M Funayama R Shirota M Nakayama K Lack of Transcription Triggers H3K27me3 Accumulation in the Gene Body Cell Rep 2016 16 696 706 27396330
  19. Jeck WR Siebold AP Sharpless NE Review: a meta-analysis of GWAS and age-associated diseases Aging Cell 2012 11 727 731 22888763 PMC3444649
  20. Kanao H Enomoto T Ueda Y Fujita M Nakashima R Ueno Y Miya-take T Yoshizaki T Buzard GS Kimura T Correlation between p14(ARF)/p16(INK4A) expression and HPV infection in uterine cervical cancer Cancer Lett 2004 213 31 37 15312681
  21. Kaneko S Son J Bonasio R Shen SS Reinberg D Nascent RNA interaction keeps PRC2 activity poised and in check Genes Dev 2014 28 1983 1988 25170018 PMC4173153
  22. Kim TK Hemberg M Gray JM Costa AM Bear DM Wu J Harmin DA Laptewicz M Barbara-Haley K Kuersten S Widespread transcription at neuronal activity-regulated enhancers Nature 2010 465 182 187 20393465 PMC3020079
  23. Kojima Y Ye J Nanda V Wang Y Flores AM Jarr KU Tsantilas P Guo L Finn AV Virmani R Leeper NJ Knockout of the murine ortholog to the human 9p21 coronary artery disease locus leads to smooth muscle cell proliferation, vascular calcification, and advanced atherosclerosis Circulation 2020 141 1274 1276 32282248 PMC7158761
  24. Larson MH Gilbert LA Wang X Lim WA Weissman JS Qi LS CRISPR interference (CRISPRi) for sequence-specific control of gene expression Nat Protoc 2013 8 2180 2196 24136345 PMC3922765
  25. Lavarone E Barbieri CM Pasini D Dissecting the role of H3K27 acetylation and methylation in PRC2 mediated control of cellular identityy Nat Commun 2019 10 1679 30976011 PMC6459869
  26. Lazorthes S Vallot C Briois S Aguirrebengoa M Thuret JY Laurent GS Rougeulle C Kapranov P Mann C Trouche D A vlincRNA participates in senescence maintenance by relieving H2AZ-mediated repression at the INK4 locus Nat Commun 2015 6 1 16 PMC4309439 25601475
  27. Li H Collado M Villasante A Strati K Ortega S Canamero M Blasco MA Serrano M The Ink4/Arf locus is a barrier for iPS cell reprogramming Nature 2009 460 1136 1139 19668188 PMC3578184
  28. Li W Notani D Ma Q Tanasa B Nunez E Chen AY Merkurjev D Zhang J Ohgi K Song X Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation Nature 2013 498 516 520 23728302 PMC3718886
  29. Liu Z Legant WR Chen BC Li L Grimm JB Lavis LD Betzig E Tjian R 3D imaging of Sox2 enhancer clusters in embryonic stem cells eLife 2014 3 e04236 25537195 PMC4381973
  30. Lo Sardo V Chubukov P Ferguson W Kumar A Teng EL Duran M Zhang L Cost G Engler AJ Urnov F Unveiling the Role of the Most Impactful Cardiovascular Risk Locus through Haplotype Editing Cell 2018 175 1796 1810 e20 30528432 PMC6346426
  31. Matheu A Maraver A Collado M Garcia-Cao I Canamero M Borras C Flores JM Klatt P Vina J Serrano M Anti-aging activity of the Ink4/Arf locus Aging Cell 2009 8 152 161 19239418
  32. McLaughlin-Drubin ME Park D Munger K Tumor suppressor p16INK4A is necessary for survival of cervical carcinoma cell lines Proc Natl Acad Sci USA 2013 110 16175 16180 24046371 PMC3791710
  33. Mikhaylichenko O Bondarenko V Harnett D Schor IE Males M Viales RR Furlong EEM The degree of enhancer or promoter activity is reflected by the levels and directionality of eRNA transcription Genes Dev 2018 32 42 57 29378788 PMC5828394
  34. Moorthy SD Davidson S Shchuka VM Singh G Malek-Gilani N Langroudi L Martchenko A So V Macpherson NN Mitchell JA Enhancers and super-enhancers have an equivalent regulatory role in embryonic stem cells through regulation of single or multiple genes Genome Res 2017 27 246 258 27895109 PMC5287230
  35. Mosteiro L Pantoja C de Martino A Serrano M Senescence promotes in vivo reprogramming through p16INK4aand IL-6 Aging Cell 2018 17 e12711 PMC5847859 29280266
  36. Mousavi K Zare H Dell’orso S Grontved L Gutierrez-Cruz G Derfoul A Hager GL Sartorelli V eRNAs promote transcription by establishing chromatin accessibility at defined genomic loci Mol Cell 2013 51 606 617 23993744 PMC3786356
  37. Muerdter F Boryń ŁM Woodfin AR Neumayr C Rath M Zabidi MA Pagani M Haberle V Kazmar T Catarino RR Resolving systematic errors in widely used enhancer activity assays in human cells Nat Methods 2018 15 141 149 29256496 PMC5793997
  38. Nicolas D Zoller B Suter DM Naef F Modulation of transcriptional burst frequency by histone acetylation Proc Natl Acad Sci USA 2018 115 7153 7158 29915087 PMC6142243
  39. Ostuni R Piccolo V Barozzi I Polletti S Termanini A Bonifacio S Curina A Prosperini E Ghisletti S Natoli G Latent enhancers activated by stimulation in differentiated cells Cell 2013 152 157 171 23332752
  40. Pauck A Lener B Hoell M Kaiser A Kaufmann AM Zwerschke W Jansen-Duürr P Depletion of the cdk inhibitor p16INK4a differentially affects proliferation of established cervical carcinoma cells J Virol 2014 88 5256 5262 24599991 PMC4019144
  41. Pnueli L Rudnizky S Yosefzon Y Melamed P RNA transcribed from a distal enhancer is required for activating the chromatin at the promoter of the gonadotropin a-subunit gene Proc Natl Acad Sci USA 2015 112 4369 4374 25810254 PMC4394321
  42. Raisner R Kharbanda S Jin L Jeng E Chan E Merchant M Haverty PM Bainer R Cheung T Arnott D Enhancer Activity Requires CBP/P300 Bromodomain-Dependent Histone H3K27 Acetylation Cell Rep 2018 24 1722 1729 30110629
  43. Rao SSP Huntley MH Durand NC Stamenova EK Bochkov ID Robinson JT Sanborn AL Machol I Omer AD Lander ES Aiden EL A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping Cell 2014 159 1665 1680 25497547 PMC5635824
  44. Samani NJ Erdmann J Hall AS Hengstenberg C Mangino M Mayer B Dixon RJ Meitinger T Braund P Wichmann H-E WTCCC and the Cardiogenics Consortium Genomewide association analysis of coronary artery disease N Engl J Med 2007 357 443 453 17634449 PMC2719290
  45. Saxena M Roman AKS O’Neill NK Sulahian R Jadhav U Shivdasani RA Transcription factor-dependent ‘anti-repressive’ mammalian enhancers exclude H3K27me3 from extended genomic domains Genes Dev 2017 31 2391 2404 29321178 PMC5795785
  46. Sherr CJ Ink4-Arf locus in cancer and aging Wiley Interdiscip Rev Dev Biol 2012 1 731 741 22960768 PMC3434949
  47. van de Werken HJ de Vree PJ Splinter E Holwerda SJ Klous P de Wit E de Laat W 4C technology: protocols and data analysis Methods Enzymol 2012 513 89 112 22929766
  48. Vazquez-Vega S Sanchez-Suarez LP Andrade-Cruz R Castellanos-Juarez E Contreras-Paredes A Lizano-Soberon M Garcia-Carranca A Benitez Bribiesca L Regulation of p14ARF expression by HPV-18 E6 variants J Med Virol 2013 85 1215 1221 23918540
  49. Visel A Zhu Y May D Afzal V Gong E Attanasio C Blow MJ Cohen JC Rubin EM Pennacchio LA Targeted deletion of the 9p21 non-coding coronary artery disease risk interval in mice Nature 2010 464 409 412 20173736 PMC2938076
  50. Wang X Paucek RD Gooding AR Brown ZZ Ge EJ Muir TW Cech TR Molecular analysis of PRC2 recruitment to DNA in chromatin and its inhibition by RNA Nat Struct Mol Biol 2017 24 1028 1038 29058709 PMC5771497
  51. Welter D MacArthur J Morales J Burdett T Hall P Junkins H Klemm A Flicek P Manolio T Hindorff L Parkinson H The NHGRI GWAS Catalog, a curated resource of SNP-trait associations Nucleic Acids Res 2014 42 D1001 D1006 24316577 PMC3965119
  52. Whyte WA Orlando DA Hnisz D Abraham BJ Lin CY Kagey MH Rahl PB Lee TI Young RA Master transcription factors and mediator establish super-enhancers at key cell identity genes Cell 2013 153 307 319 23582322 PMC3653129
  53. Wu H Nord AS Akiyama JA Shoukry M Afzal V Rubin EM Pennacchio LA Visel A Tissue-specific RNA expression marks distantacting developmental enhancers PLoS Genet 2014 10 e1004610 25188404 PMC4154669
  54. Yang J Kantrow S Sai J Hawkins OE Boothby M Ayers GD Young ED Demicco EG Lazar AJ Lev D Richmond A INK4a/ARF [corrected] inactivation with activation of the NF-κB/IL-6 pathway is sufficient to drive the development and growth of angiosarcoma Cancer Res 2012 72 4682 4695 22836752 PMC3459578
  55. Yap KL Li S Munoz-Cabello AM Raguz S Zeng L Mujtaba S Gil J Walsh MJ Zhou MM Molecular interplay of the noncoding RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a Mol Cell 2010 38 662 674 20541999 PMC2886305
  56. Zhang Y Xiong Y Yarbrough WG ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways Cell 1998 92 725 734 9529249
  57. Zhang CY Bao W Wang LH Downregulation of p16(ink4a) inhibits cell proliferation and induces G1 cell cycle arrest in cervical cancer cells Int J Mol Med 2014 33 1577 1585 24714974
  58. Zhang Y Hyle J Wright S Shao Y Zhao X Zhang H Li C A cis-element within the ARF locus mediates repression of p16INK4A expression via long-range chromatin interactions Proc Natl Acad Sci USA 2019 116 26644 26652 PMC6936709 31818950
  59. Zhang T Zhang Z Dong Q Xiong J Zhu B Histone H3K27 acetylation is dispensable for enhancer activity in mouse embryonic stem cells Genome Biol 2020 21 45 32085783 PMC7035716
  60. Zhao Y Zhou J He L Li Y Yuan J Sun K Chen X Bao X Esteban MA Sun H Wang H MyoD induced enhancer RNA interacts with hnRNPL to activate target gene transcription during myogenic differentiation Nat Commun 2019 10 5787 31857580 PMC6923398
  61. Zhou X Han X Wittfeldt A Sun J Liu C Wang X Gan LM Cao H Liang Z Long non-coding RNAANRIL regulates inflammatory responses as a novel component of NF-kB pathway RNA Biol 2016 13 98 108 26618242 PMC4829310

Cited by

Sorted by citations

Authors
Farooq, U., Saravanan, B., Islam, Z., Walavalkar, K., Singh, A. K., Jayani, R. S., Meel, S., Swaminathan, S., & Notani, D.
Affiliations
Tata Institute of Fundamental Research (IN)
Corresponding author
Notani, Dimple
ORCID
Venue
Cell Reports
Publisher
Elsevier BV
Published
2021-03
Volume
34
Issue
12
Pages
108898
ISSN
2211-1247 (ISSN-L)
DOI
10.1016/j.celrep.2021.108898
PMC
PMC7611173
OpenAlex
W3138899071
Cited by
29
References
6
Citations by year
2026: 5 · 2025: 3 · 2024: 3 · 2023: 10 · 2022: 2 · 2021: 6
FWCI
1.65
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Reports (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion, cc-by-nc-nd)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Tata Institute of Fundamental Research
  • Department of Atomic Energy, Government of India (12-R, D-TFR-5.04-0800)
  • National Centre for Biological Sciences
  • Wellcome Trust DBT India Alliance (IA/1/14/2/501539)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chromatin Remodeling and Cancer
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer-related molecular mechanisms research
Keywords
INK4a/ARF locus, enhancer clusters, enhancer-promoter interactions, EZH2, H3K27me3, ANRIL, 9p21, GWAS loci, long-range transcriptional regulation, enhancer deletion
MeSH terms
Enhancer of Zeste Homolog 2 Protein, Chromosomes, Human, Pair 9, Enhancer Elements, Genetic, Gene Expression Regulation, HeLa Cells, Humans, Promoter Regions, Genetic, Transcription, Genetic, Cyclin-Dependent Kinase Inhibitor p16, Genetic Loci, HEK293 Cells

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