Modeling the effect of prolonged ethanol exposure on global gene expression and chromatin accessibility in normal 3D colon organoids

Autoři: Matthew Devall aff001;  Lucas T. Jennelle aff001;  Jennifer Bryant aff001;  Stephanie Bien aff002;  Ulrike Peters aff002;  Steven Powell aff003;  Graham Casey aff001
Působiště autorů: Center for Public Health Genomics, Department of Public Health Sciences, University of Virginia, Charlottesville, Virginia, United States of America aff001;  Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America aff002;  Digestive Health Center, Gastroenterology and Heaptology, University of Virginia, Charlottesville, Virginia, United States of America aff003
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: 10.1371/journal.pone.0227116


In this study we aimed to explore the potential biological effect of ethanol exposure on healthy colon epithelial cells using normal human colon 3D organoid “mini-gut” cultures. In numerous published studies ethanol use has been shown to be an environmental risk factor for colorectal cancer (CRC) development; however, the influence of ethanol exposure on normal colon epithelial cell biology remains poorly understood. We investigated the potential molecular effects of ethanol exposure in normal colon 3D organoids in a small pilot study (n = 3) using RNA-seq and ATAC-seq. We identify 1965 differentially expressed genes and 2217 differentially accessible regions of chromatin in response to ethanol treatment. Further, by cross-referencing our results with previously published analysis in colorectal cancer cell lines, we have not only validated a number of reported differentially expressed genes, but also identified several novel candidates for future investigation. In summary, our data highlights the potential importance for the use of normal colon 3D organoid models as a novel tool for the investigation of the relationship between the effects of environmental risk factors associated with colorectal cancer and the molecular mechanisms through which they confer this risk.

Klíčová slova:

Colon – Colorectal cancer – Ethanol – Gene expression – Genomic libraries – Chromatin – Organoids – RNA isolation


1. Chen W, Frankel WL. A practical guide to biomarkers for the evaluation of colorectal cancer. Mod Pathol. 2019.

2. Huyghe JR, Bien SA, Harrison TA, Kang HM, Chen S, Schmit SL, et al. Discovery of common and rare genetic risk variants for colorectal cancer. Nat Genet. 2019;51[1]:76–87. doi: 10.1038/s41588-018-0286-6 30510241

3. Bishehsari F, Mahdavinia M, Vacca M, Malekzadeh R, Mariani-Costantini R. Epidemiological transition of colorectal cancer in developing countries: environmental factors, molecular pathways, and opportunities for prevention. World J Gastroenterol. 2014;20[20]:6055–72. doi: 10.3748/wjg.v20.i20.6055 24876728

4. Allemani C, Weir HK, Carreira H, Harewood R, Spika D, Wang XS, et al. Global surveillance of cancer survival 1995–2009: analysis of individual data for 25,676,887 patients from 279 population-based registries in 67 countries (CONCORD-2). Lancet. 2015;385(9972):977–1010. doi: 10.1016/S0140-6736(14)62038-9 25467588

5. Roerink SF, Sasaki N, Lee-Six H, Young MD, Alexandrov LB, Behjati S, et al. Intra-tumour diversification in colorectal cancer at the single-cell level. Nature. 2018;556(7702):457-+.

6. Dame MK, Attili D, McClintock SD, Dedhia PH, Ouillette P, Hardt O, et al. Identification, isolation and characterization of human LGR5-positive colon adenoma cells. Development. 2018;145[6].

7. Clevers H. Modeling Development and Disease with Organoids. Cell. 2016;165[7]:1586–97. doi: 10.1016/j.cell.2016.05.082 27315476

8. Lindeboom RG, van Voorthuijsen L, Oost KC, Rodriguez-Colman MJ, Luna-Velez MV, Furlan C, et al. Integrative multi-omics analysis of intestinal organoid differentiation. Mol Syst Biol. 2018;14[6]:e8227. doi: 10.15252/msb.20188227 29945941

9. Fujii M, Matano M, Toshimitsu K, Takano A, Mikami Y, Nishikori S, et al. Human Intestinal Organoids Maintain Self-Renewal Capacity and Cellular Diversity in Niche-Inspired Culture Condition. Cell Stem Cell. 2018;23[6]:787–93 e6.

10. Beyaz S, Mana MD, Roper J, Kedrin D, Saadatpour A, Hong SJ, et al. High-fat diet enhances stemness and tumorigenicity of intestinal progenitors. Nature. 2016;531(7592):53–8. doi: 10.1038/nature17173 26935695

11. Lister R, O'Malley RC, Tonti-Filippini J, Gregory BD, Berry CC, Millar AH, et al. Highly integrated single-base resolution maps of the epigenome in Arabidopsis. Cell. 2008;133[3]:523–36. doi: 10.1016/j.cell.2008.03.029 18423832

12. Byron SA, Van Keuren-Jensen KR, Engelthaler DM, Carpten JD, Craig DW. Translating RNA sequencing into clinical diagnostics: opportunities and challenges. Nat Rev Genet. 2016;17[5]:257–71. doi: 10.1038/nrg.2016.10 26996076

13. Buenrostro JD, Giresi PG, Zaba LC, Chang HY, Greenleaf WJ. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat Methods. 2013;10[12]:1213–8. doi: 10.1038/nmeth.2688 24097267

14. Wang J, Zibetti C, Shang P, Sripathi SR, Zhang PW, Cano M, et al. ATAC-Seq analysis reveals a widespread decrease of chromatin accessibility in age-related macular degeneration. Nat Commun. 2018;9. doi: 10.1038/s41467-017-01881-x

15. Cao J, Cusanovich DA, Ramani V, Aghamirzaie D, Pliner HA, Hill AJ, et al. Joint profiling of chromatin accessibility and gene expression in thousands of single cells. Science. 2018.

16. Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature. 2009;459(7244):262–U147. doi: 10.1038/nature07935 19329995

17. Forsyth CB, Tang Y, Shaikh M, Zhang L, Keshavarzian A. Alcohol stimulates activation of Snail, epidermal growth factor receptor signaling, and biomarkers of epithelial-mesenchymal transition in colon and breast cancer cells. Alcohol Clin Exp Res. 2010;34[1]:19–31. doi: 10.1111/j.1530-0277.2009.01061.x 19860811

18. Wang S, Xu M, Li F, Wang X, Bower KA, Frank JA, et al. Ethanol promotes mammary tumor growth and angiogenesis: the involvement of chemoattractant factor MCP-1. Breast Cancer Res Treat. 2012;133[3]:1037–48. doi: 10.1007/s10549-011-1902-7 22160640

19. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29[1]:15–21. doi: 10.1093/bioinformatics/bts635 23104886

20. Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30[7]:923–30. doi: 10.1093/bioinformatics/btt656 24227677

21. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15[12]:550. doi: 10.1186/s13059-014-0550-8 25516281

22. Corces MR, Trevino AE, Hamilton EG, Greenside PG, Sinnott-Armstrong NA, Vesuna S, et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat Methods. 2017;14[10]:959–62. doi: 10.1038/nmeth.4396 28846090

23. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nature Methods. 2012;9[4]:357–U54. doi: 10.1038/nmeth.1923 22388286

24. Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE, et al. Model-based Analysis of ChIP-Seq (MACS). Genome Biology. 2008;9(9).

25. Stark R and Brown G. DiffBind: differential binding analysis of ChIP-Seq peak data: Bioconductor; 2011.

26. Chen J, Bardes EE, Aronow BJ, Jegga AG. ToppGene Suite for gene list enrichment analysis and candidate gene prioritization. Nucleic Acids Res. 2009;37(Web Server issue):W305–11. doi: 10.1093/nar/gkp427 19465376

27. Rohart F, Gautier B, Singh A, Le Cao KA. mixOmics: An R package for 'omics feature selection and multiple data integration. Plos Comput Biol. 2017;13[11].

28. Forsyth CB, Tang YM, Shaikh M, Zhang LJ, Keshavarzian A. Alcohol Stimulates Activation of Snail, Epidermal Growth Factor Receptor Signaling, and Biomarkers of Epithelial-Mesenchymal Transition in Colon and Breast Cancer Cells. Alcohol Clin Exp Res. 2010;34[1]:19–31. doi: 10.1111/j.1530-0277.2009.01061.x 19860811

29. Forsyth CB, Shaikh M, Bishehsari F, Swanson G, Voigt RM, Dodiya H, et al. Alcohol Feeding in Mice Promotes Colonic Hyperpermeability and Changes in Colonic Organoid Stem Cell Fate. Alcohol Clin Exp Res. 2017;41[12]:2100–13. doi: 10.1111/acer.13519 28992396

30. Ackermann AM, Wang Z, Schug J, Naji A, Kaestner KH. Integration of ATAC-seq and RNA-seq identifies human alpha cell and beta cell signature genes. Mol Metab. 2016;5[3]:233–44. doi: 10.1016/j.molmet.2016.01.002 26977395

31. Jaiswal M, Dvorsky R, Amin E, Risse SL, Fansa EK, Zhang SC, et al. Functional Cross-talk between Ras and Rho Pathways A Ras-SPECIFIC GTPase-ACTIVATING PROTEIN (p120RasGAP) COMPETITIVELY INHIBITS THE RhoGAP ACTIVITY OF DELETED IN LIVER CANCER (DLC) TUMOR SUPPRESSOR BY MASKING THE CATALYTIC ARGININE FINGER. J Biol Chem. 2014;289[10]:6839–49. doi: 10.1074/jbc.M113.527655 24443565

32. Saeed O, Lopez-Beltran A, Fisher KW, Scarpelli M, Montironi R, Cimadamore A, et al. RAS genes in colorectal carcinoma: pathogenesis, testing guidelines and treatment implications. J Clin Pathol. 2019;72[2]:135–9. doi: 10.1136/jclinpath-2018-205471 30425122

33. Moon JW, Lee SK, Lee YW, Lee JO, Kim N, Lee HJ, et al. Alcohol induces cell proliferation via hypermethylation of ADHFE1 in colorectal cancer cells. Bmc Cancer. 2014;14. doi: 10.1186/1471-2407-14-14

Článek vyšel v časopise


2020 Číslo 1