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dc.contributor.author
Benzinger, Dirk
dc.contributor.supervisor
Khammash, Mustafa Hani
dc.contributor.supervisor
Ackermann, Martin
dc.contributor.supervisor
Pelet, Serge
dc.date.accessioned
2019-08-22T06:56:44Z
dc.date.available
2019-08-21T14:58:33Z
dc.date.available
2019-08-22T06:53:45Z
dc.date.available
2019-08-22T06:56:44Z
dc.date.issued
2019
dc.identifier.uri
http://hdl.handle.net/20.500.11850/359761
dc.identifier.doi
10.3929/ethz-b-000359761
dc.description.abstract
Gene expression can exhibit substantial cell-to-cell variability, which partly stems from stochasticity inherent to the transcription process. Nevertheless, its precise regulation is crucial for most biological processes as well as in biotechnological applications. Many of the steps involved in transcriptional regulation are highly dynamic. For example, a variety of transcription factors (TFs) exhibit a pulsatile pattern of activity. However, the complexity of signal transduction and gene regulation hampers our ability to analyze how the dynamic activity of TFs affects transcription and cellular heterogeneity. In this thesis, we establish a synthetic biology approach that makes use of a fast-acting, light-responsive (optogenetic) TF to quantitatively study multiple aspects of transcriptional regulation. To this end, we first implement and thoroughly characterize an optogenetic gene expression system based on the synthetic TF VP-EL222 in S. cerevisiae (chapter 2). We then compare how constant and pulsatile input signals affect gene expression. We find that pulse-width-modulation (PWM), meaning that the duration of input pulses is modulated to regulate gene expression levels, results in the coordinated expression of genes whose promoters respond differentially to constant input signals. We further show that pulsatile TF regulation can reduce cell-to-cell variability in protein expression and that expression mean and variability can be independently tuned by adjusting the frequency of input signals. This phenomenon is then employed to quantify the phenotypic consequence of cell-to-cell variability in metabolic enzyme expression. Both mathematical modeling and experiments indicate that the observed variability reduction largely stems from the attenuation of heterogeneity arising from cell-to-cell differences in TF expression. Modeling further suggests that pulsatile inputs may reduce intrinsic variability that arises from the dynamic/bursty nature of transcription. We then combine the optogenetic TF with live-cell quantification of nascent RNA in order to study transcriptional regulation in more detail (chapter 3). We find that transcription in fact occurs in discontinuous bursts whose duration and timing are modulated by TF activity. By probing the system with pulsed TF inputs, we uncover that promoter activation is largely memoryless and that bursts are terminated upon TF unbinding. Based on these results, we propose a mechanistic model of transcriptional bursting based on TF binding and a rate limiting step, which we interpret as chromatin remodeling, that quantitatively reproduces our experimental observations. Our results demonstrate the merit of using easily controllable synthetic systems to gain new insight into fundamental biological processes. The knowledge gained by this approach may be applied to improve gene expression regulation in biotechnological and biomedical applications.
en_US
dc.format
application/pdf
en_US
dc.language.iso
en
en_US
dc.publisher
ETH Zurich
en_US
dc.rights.uri
http://creativecommons.org/licenses/by-nc-nd/4.0/
dc.title
Optogenetic Regulation and Interrogation of Gene Expression Variability
en_US
dc.type
Doctoral Thesis
dc.rights.license
Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International
dc.date.published
2019-08-22
ethz.size
182 p.
en_US
ethz.code.ddc
DDC - DDC::5 - Science::570 - Life sciences
ethz.identifier.diss
25770
en_US
ethz.publication.place
Zurich
en_US
ethz.publication.status
published
en_US
ethz.leitzahl
ETH Zürich::00002 - ETH Zürich::00012 - Lehre und Forschung::00007 - Departemente::02060 - Dep. Biosysteme / Dep. of Biosystems Science and Eng.::03921 - Khammash, Mustafa / Khammash, Mustafa
en_US
ethz.date.deposited
2019-08-21T14:58:45Z
ethz.source
FORM
ethz.eth
yes
en_US
ethz.availability
Open access
en_US
ethz.rosetta.installDate
2019-08-22T06:54:13Z
ethz.rosetta.lastUpdated
2019-08-22T06:57:12Z
ethz.rosetta.versionExported
true
ethz.COinS
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