Role of Chromatin Associated RNAi Components in Gene Expression Regulation in Mammalian Cells

  • Bodor Fallatah

Student thesis: Doctoral Thesis

Abstract

RNA interference (RNAi) is an important pathway that regulates gene expression in several organisms. The role of RNAi in post-transcriptional gene silencing in the cytoplasm is well characterized. In contrast, the role of RNAi components in the nucleus remains to be elucidated. Previous reports have indicated that RNAi components (Dicer and Argonaute proteins) and small RNAs act in the nucleus to regulate various pathways including heterochromatin formation, transposable elements repression, RNA Pol II processivity and alternative splicing. Nuclear Ago1 and Dicer have also been found to associate with active promoters and enhancers in mammalian cells, however their functional roles and mechanisms remain elusive. In this work, I investigated the functional role of nuclear RNAi components in gene expression regulation during skeletal muscle differentiation. To address this question, I undertook genomic and biochemical approaches applied to myogenic cells (C2C12) as a model system. I found that Ago1 and Dicer are present in the nucleus of C2C12 cells and expressed during differentiation. Chromatin Immunoprecipitation (ChIP) coupled with high throughput sequencing and quantitative real-time PCR indicate that Ago1 and Dicer are enriched at promoters and enhancer regions of myogenic genes. Interestingly, I found that depletion of Ago1 and Dicer reduces enhancer RNAs (eRNAs) levels at enhancer regions and expression of MyoD during differentiation. I observed that loss of Ago1 impacts differentiation, whereas, loss of Dicer leads to cell death and has severe effects on C2C12 cells. Moreover, using Chromosome Conformation Capture (3C), I revealed that Ago1 is involved in enhancer-promoter interaction at MyoD locus. The knockdown of Ago1 destabilizes these interactions and decreases the expression of MyoD. Finally, I demonstrated that Ago1 binds to eRNAs and interacts with CBP Acetyl-transferase in the nucleus of myotube cells. Ago1 depletion leads to loss of eRNA-CBP interaction and consequent impairment of CBP acetyltransferase activity and failure of MyoD mediated activation of the myogenic program. Taken together, these finding indicate that nuclear Ago1 together with eRNAs and CBP regulates MyoD expression by stimulating histone acetylation during differentiation. This study uncovered a novel function of chromatin associated Ago1 in gene expression regulation during mammalian skeletal muscle differentiation.
Date of AwardApr 2019
Original languageEnglish (US)
Awarding Institution
  • Biological, Environmental Science and Engineering
SupervisorValerio Orlando (Supervisor)

Keywords

  • RNA interference
  • Gene Expression Regulation
  • Epigenetic Regulation
  • Skeletal muscle differentiation
  • Non-coding RNA
  • Mammalian Cells

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