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University of Bayreuth and Elite Network of Bavaria
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Changes in the transcriptional programs of neurons or neuronal progenitors lie at the heart of neuronal plasticity and neurogenesis, respectively. Genes that regulate these processes are under the control of enhancer elements that are differentially regulated during neuronal stimulation and brain development. Enhancers are DNA sequences bound by at times multiple transcription factors. In a combinatorial activation process the combined activity of all bound factors is believed to determine the extent of target gene activation.
In addition, neuronal enhancers were recently shown to be actively transcribed, giving rise to non-coding enhancer RNAs (eRNAs). eRNAs significantly contribute to the activation of most immediate early genes (IEG) in mouse neurons. Moreover, eRNA expression levels serve as a better indicator of enhancer activity than transcription factor binding data. However, how enhancer RNAs are able to direct brain development and neuronal plasticity in vivo is unknown. In particular, it is unknown how much enhancer transcription contributes to enhancer function during these processes.
We pursue the hypothesis that enhancer RNAs directly influence the Pol II transcription machinery by diminishing promoter-proximal pausing of important neuronal genes. To examine this hypothesis, we will combine in vitro biochemistry, next-generation sequencing and in vivo studies of zebrafish neurogenesis. Specifically, we will address the following questions:
1. Are neuronal enhancer RNAs able to abrogate promoter-proximal pausing in vitro?
2. Are neuronal enhancer RNAs conserved between zebrafish and mouse?
3. What is the role of enhancer RNAs in zebrafish nervous system development?
Zebrafish is a uniquely suited model system to study eRNA function on an organismic level. It combines the ease of being easily able to image neurogenesis with the ability to manipulate eRNA functionality using both antisense techniques and CRISPRi. Despite the fact that most emerging classes of non-coding RNA (ncRNA) influence gene expression on the transcriptional level there is very little insight to date on the underlying mechanisms. This proposal bridges this gap by directly integrating a mechanistic study on Pol II transcription elongation with functional analyses of zebrafish neurogenesis in vivo. To the best of our knowledge there are no reports to date that have characterized the impact of enhancer RNAs on nervous system development and brain function on an organismic level.
Claus-D. Kuhn, Jeremy E. Wilusz, Yuxuan Zheng, Peter A. Beal, and Leemor Joshua-Tor (2015). On-Enzyme Refolding Permits Small RNA and tRNA Surveillance by the CCA-Adding Enzyme. Cell 160, 1-15.
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