Mesane gen regülasyonunda etkili süper-enhancer'ların belirlenmesi

Year 2020, Volume: 77 Issue: 1, 41 - 50, 01.03.2020

Serap Erkek

Abstract

Amaç: Gen ekspresyonunun düzgün bir şekilde gerçekleşmesinde hızlandırıcı enhancer adı verilen genomik bölgeler, transkripsiyon faktörlerini genlerin promotor bölgeleri ile bir araya getirir. Enhancer bölgeleri genomda histon H3 lizin 27 asetilasyon H3K27ac sinyaline sahip olmalarıyla tanımlanabilmektedir. Süperenhancer’lar genomda yüksek H3K27ac sinyaline sahip birkaç enhancer bölgesinin kümelenmesiyle oluşan regülatör bölgelerdir. Süper-enhancer’ların hücre-spesifik gen ekspresyon profillerinin düzenlemesinde önemli bir rol oynadığı gösterilmiştir. Bu çalışmanın amacı, Roadmap Epigenom konsorsiyum kapsamında oluşturulan mesane dokusu H3K27ac kromatin immünopresipitasyon dizileme ChIP-seq verisinin analiz edilerek mesane dokusunu regüle eden süper-enhancer’ların tanımlanması ve bağlantılı oldukları genlerin ortaya çıkarılmasıdır. Yöntem: Mesane H3K27ac ChIP-seq verisine Roadmap Epigenom veritabanından erişilmiştir. Veri Homer ChIP-seq analiz platformunda analiz edilmiştir. Süper-enhancer’lar Homer platformunda ‘zirve bulma’ fonksiyonunu findPeaks ‘super’ modunda kullanılarak belirlenmiştir. Belirlenen süper-enhancer’ların genlerle ilişkilendirilmesi Homer ‘zirve bölge anlamlandırma’ fonksiyonu annotatePeaks kullanılarak yapılmıştır. H3K27ac sinyalinin ve süper-enhancer bölgelerinin görsel olarak sunumu için UCSC GenomeBrowser altyapısından faydalanılmıştır. Süper-enhancer bağlantılı genlerin etkileşim ağlarını ve rol oynadıkları sinyal yolaklarını bulmak için STRING protein etkileşim veritabanı kullanılmıştır. Bulgular: Mesane dokusunda 602 süper-enhancer belirlenmiştir. İlk en yüksek 100 H3K27ac sinyal değerine sahip olan süper-enhancer’lar aralarında TBX3, RARA, RXRA, RASSF1, DAB2IP’nin olduğu genlerle ilişkilendirilmiştir. Süper-enhancer-regüle olan süperenhancer’ın gen transkripsiyon başlangıç nokatısına uzaklığı max 10 kilobaz genlerin n=386 organ gelişimi, epitel hücre farklılaşması, retinoik asit metabolizmasında görev aldıkları belirlenmiştir. Aynı zamanda mesane süper-enhancer’ları ile ilişkili olan genlerin istatistiksel olarak anlamlı bir biçimde yanlış bulgu oranı = 1.49e-07 transkripsiyon faktör görevini yapan protein grubunda yer aldığı ve etkileşim ağı oluşturdukları belirlenmiştir. Sonuç: Bu çalışmayla mesane dokusunu regüle eden süper-enhancer regülatör bölgeler ortaya çıkarılmıştır. İlişkili genlerin normal mesane yapısının korunmasındaki rolleri ve söz konusu genlerin mesane kanser mekanizmalarıyla bağlantıları göz önünde bulundurularak, elde edilen sonuçların mesane biyolojisinin daha iyi anlaşılmasına katkı sağladığı düşünülmektedir.

Keywords

Transkripsiyon, mesane, histon, hızlandırıcı (enhancer)

Identification of the super-enhancers involved in bladder gene regulation

Abstract

Objective: Enhancer elements in the genome take a role in establishment of proper gene expression patterns via bringing transcription factors together with the promoter regions of the genes. Acetylation of lysine K27 on histone H3 H3K27ac marks the enhancer regions in the genome. Super-enhancers are regulatory regions which have unusual high signal of H3K27ac and consist of several enhancer elements. Super-enhancers play a critical role in setting of correct cell-type specific gene expression programs. The aim of this study is to identify the super-enhancers characterizing normal bladder and associate these super-enhancers with the genes via analyzing bladder H3K27ac chromatin immunoprecipitation sequencing ChIP-seq data generated within Roadmap Epigenomics Project. Methods: Bladder H3K27ac ChIP-seq data was downloaded from Roadmap Epigenomics server. The data was analyzed on Homer ChIP-seq analysis platform. Super-enhancers were called using ‘findPeaks’ function of the platform in ‘super’ mode. The identified super-enhancers were associated with the genes using ‘annotatePeaks’ function of Homer platform. The visualization of H3K27ac ChIP-seq signal and identified super-enhancers was performed using UCSC GenomeBrowser. The protein-protein interaction networks of super-enhancer regulated genes and the pathways involved were determined using STRING protein interaction database. Results: 602 super-enhancers was identified in bladder tissue. Among the genes which were associated with super-enhancers having the top 100 highest H3K27ac signal were TBX3, RARA, RXRA, RASSF1, DAB2IP. Super-enhancer regulated max distance of the super-enhancer to transcriptional start site of gene is 10 kb genes n=386 were identified to be involved in organ development, epithelial cell differentiation, and retinoic acid metabolism. In addition, it was determined that transcription factors were significantly False Discovery Rate FDR =1.49e07 enriched among the genes regulated by superenhancers and those genes had a significant proteinprotein interaction. Conclusion: With this study, the super-enhancers regulating normal bladder were determined. Given the fact that associated genes are involved in the maintenance of normal bladder homeostasis and the misregulation same set of genes take a role in bladder cancer, it is estimated that the results obtained with this study will largely contribute to a better understanding of bladder biology

Keywords

Transcription, bladder, histone, enhancer

References

• 1. Niederriter AR, Varshney A, Parker SC, Martin DM. Super Enhancers in Cancers, Complex Disease, and Developmental Disorders. Genes (Basel). 2015;6(4):1183-200.
• 2. Banerji J, Rusconi S, Schaffner W. Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences. Cell, 1981;27(2 Pt 1):299-308.
• 3. Pott S, Lieb JD. What are super-enhancers? Nat Genet. 2015;47(1):8-12.
• 4. Shendure J, Balasubramanian S, Church GM, Gilbert W, Rogers J, Schloss JA, et al. DNA sequencing at 40: past, present and future. Nature, 2017;550(7676):345-53.
• 5. Morange M. The relations between genetics and epigenetics: a historical point of view. Ann N Y Acad Sci, 2002;981:50-60.
• 6. Milne TA, Zhao K, Hess JL. Chromatin immunoprecipitation (ChIP) for analysis of histone modifications and chromatin-associated proteins. Methods Mol Biol, 2009;538:409-23.
• 7. Consortium EP. An integrated encyclopedia of DNA elements in the human genome. Nature. 2012;489(7414):57-74.
• 8. Roadmap Epigenomics C, Kundaje A, Meuleman W, Ernst J, Bilenky M, Yen A, et al. Integrative analysis of 111 reference human epigenomes. Nature, 2015;518(7539):317-30.
• 9. Johann PD, Erkek S, Zapatka M, Kerl K, Buchhalter I, Hovestadt V, et al. Atypical teratoid/rhabdoid tumors are comprised of three epigenetic subgroups with distinct enhancer landscapes. cancer cell, 2016;29(3):379-93.
• 10. Lin CY, Erkek S, Tong Y, Yin L, Federation AJ, Zapatka M, et al. Active medulloblastoma enhancers reveal subgroup-specific cellular origins. Nature, 2016;530(7588):57-62.
• 11. Whyte WA, Orlando DA, Hnisz D, Abraham BJ, Lin CY, Kagey MH, et al. Master transcription factors and mediator establish super-enhancers at key cell identity genes. Cell, 2013;153(2):307-19.
• 12. Hnisz D, Abraham BJ, Lee TI, Lau A, Saint-Andre V, Sigova AA, et al. Super-enhancers in the control of cell identity and disease. Cell, 2013;155(4):934- 47.
• 13. Heinz S, Benner C, Spann N, Bertolino E, Lin YC, Laslo P, et al. Simple combinations of lineagedetermining transcription factors prime cisregulatory elements required for macrophage and B cell identities. Mol Cell, 2010;38(4):576-89.
• 14. Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, et al. The human genome browser at UCSC. Genome Res, 2002;12(6):996-1006.
• 15. Szklarczyk D, Franceschini A, Wyder S, Forslund K, Heller D, Huerta-Cepas J, et al. STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res, 2015;43(Database issue):D447-52.
• 16. Lan HY, Chung AC. TGF-β/Smad signaling in kidney disease. Semin Nephrol, 2012; 32(3):236-43.
• 17. Nackiewicz D, Dey P, Szczerba B, Mohammad S, Kaplan JL, McNamara CA, Deshmukh US, Bagavant H. Inhibitor of differentiation 3, a transcription factor, regulates hyperlipidemia-associated kidney disease. Nephron Exp Nephrol, 2014; 126(3):141- 7.
• 18. Mendelsohn C, Batourina E, Fung S, Gilbert T, Dodd J. Stromal cells mediate retinoid-dependent functions essential for renal development. Development, 1999;126(6):1139-48.
• 19. Thiagarajan RD, Georgas KM, Rumballe BA, Lesieur E, Chiu HS, Taylor D, et al. Identification of anchor genes during kidney development defines ontological relationships, molecular subcompartments and regulatory pathways. PLoS One, 2011;6(2):e17286.
• 20. Sharp T, Wang J, Li X, Cao H, Gao S, Moreno M, et al. A pituitary homeobox 2 (Pitx2):microRNA-200a3p:beta-catenin pathway converts mesenchymal cells to amelogenin-expressing dental epithelial cells. J Biol Chem, 2014;289(39):27327-41.
• 21. Keijzer R, van Tuyl M, Meijers C, Post M, Tibboel D, Grosveld F, et al. The transcription factor GATA6 is essential for branching morphogenesis and epithelial cell differentiation during fetal pulmonary development. Development, 2001;128(4):503-11.
• 22. Loven J, Hoke HA, Lin CY, Lau A, Orlando DA, Vakoc CR, et al. Selective inhibition of tumor oncogenes by disruption of super-enhancers. Cell, 2013;153(2):320-34.
• 23. Kispert A. T-box genes in the kidney and urinary tract. Curr Top Dev Biol, 2017;122:245-78.
• 24. Ito A, Asamoto M, Hokaiwado N, Takahashi S, Shirai T. Tbx3 expression is related to apoptosis and cell proliferation in rat bladder both hyperplastic epithelial cells and carcinoma cells. Cancer Lett.,2005;219(1):105-12.
• 25. Beukers W, Kandimalla R, Masius RG, Vermeij M, Kranse R, van Leenders GJ, et al. Stratification based on methylation of TBX2 and TBX3 into three molecular grades predicts progression in patients with pTa-bladder cancer. Mod Pathol, 2015;28(4):515-22.
• 26. Balmer JE, Blomhoff R. Gene expression regulation by retinoic acid. J Lipid Res, 2002;43(11):1773- 808.
• 27. Wang C, Ross WT, Mysorekar IU. Urothelial generation and regeneration in development, injury, and cancer. Dev Dyn, 2017;246(4):336-43.
• 28. DeGraff DJ, Cates JM, Mauney JR, Clark PE, Matusik RJ, Adam RM. When urothelial differentiation pathways go wrong: implications for bladder cancer development and progression. Urol Oncol, 2013;31(6):802-11.
• 29. Hurst RE, Waliszewski P, Waliszewska M, Bonner RB, Benbrook DM, Dar A, et al. Complexity, retinoid-responsive gene networks, and bladder carcinogenesis. Adv Exp Med Biol, 1999;462:449- 67.
• 30. Zou C, Liebert M, Zou C, Grossman HB, Lotan R. Identification of effective retinoids for inhibiting growth and inducing apoptosis in bladder cancer cells. J Urol, 2001;165(3):986-92.
• 31. Zou C, Ramakumar S, Qian L, Zou C, Zang R, Wang J, et al. Effect of retinoic acid and interferon alpha-2a on transitional cell carcinoma of bladder. J Urol. 2005;173(1):247-51.
• 32. Allen NP, Donninger H, Vos MD, Eckfeld K, Hesson L, Gordon L, et al. RASSF6 is a novel member of the RASSF family of tumor suppressors. Oncogene, 2007;26(42):6203-11.
• 33. Ha YS, Jeong P, Kim JS, Kwon WA, Kim IY, Yun SJ, et al. Tumorigenic and prognostic significance of RASSF1A expression in low-grade (WHO grade 1 and grade 2) nonmuscle-invasive bladder cancer. Urology, 2012;79(6):1411 e1-6.
• 34. Gao T, Wang S, He B, Pan Y, Song G, Gu L, et al. The association of RAS association domain family Protein1A (RASSF1A) methylation states and bladder cancer risk: a systematic review and meta-analysis. PLoS One, 2012;7(11):e48300.
• 35. Shen YJ, Kong ZL, Wan FN, Wang HK, Bian XJ, Gan HL, et al. Downregulation of DAB2IP results in cell proliferation and invasion and contributes to unfavorable outcomes in bladder cancer. Cancer Sci, 2014;105(6):704-12.
• 36. Jou YC, Tsai YS, Chen SY, Hsieh HY, Tsai HT, Tzai TS. Loss of DAB2IP expression in human urothelial carcinoma is associated with poorer recurrencefree survival. Virchows Arch, 2016;468(6):733-40.