Mesane gen regülasyonunda etkili süper-enhancer'ların belirlenmesi

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.

Anahtar Kelimeler:

Transkripsiyon, mesane, histon, hızlandırıcı (enhancer)

Identification of the super-enhancers involved in bladder gene regulation

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,

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