The combinatorics of transcriptional regulation

The ability to regulate gene expression is of central importance for the adaptability of living organisms to changes in their internal and external environment. At the transcriptional level, binding of transcription factors (TFs) in the vicinity of promoters can modulate the rate at which transcripts are produced, and as such play an important role in gene regulation. TFs with regulatory action at multiple promoters is the rule rather than the exception, with examples ranging from TFs like the cAMP receptor protein (CRP) in E. coli that regulates hundreds of different genes, to situations involving multiple copies of the same gene, such as on plasmids, or viral DNA. When the number of TFs heavily exceeds the number of binding sites, TF binding to each promoter can be regarded as independent. However, when the number of TF molecules is comparable to the number of binding sites, TF titration will result in coupling ("entanglement") between transcription of different genes. The last few decades have seen rapid advances in our ability to quantitatively measure such effects, which calls for biophysical models to explain these data. Here we develop a statistical mechanical model which takes the TF titration effect into account and use it to predict both the level of gene expression and the resulting correlation in transcription rates for a general set of promoters. To test these predictions experimentally, we create genetic constructs with known TF copy number, binding site affinities, and gene copy number; hence avoiding the need to use free fit parameters. Our results clearly prove the TF titration effect and that the statistical mechanical model can accurately predict the fold change in gene expression for the studied cases. We also generalize these experimental efforts to cover systems with multiple different genes, using the method of mRNA fluorescence in situ hybridization (FISH). Interestingly, we can use the TF titration affect as a tool to measure the plasmid copy number at different points in the cell cycle, as well as the plasmid copy number variance. Finally, we investigate the strategies of transcriptional regulation used in a real organism by analyzing the thousands of known regulatory interactions in E. coli. We introduce a "random promoter architecture model" to identify overrepresented regulatory strategies, such as TF pairs which coregulate the same genes more frequently than would be expected by chance, indicating a related biological function. Furthermore, we investigate whether promoter architecture has a systematic effect on gene expression by linking the regulatory data of E. coli to genome-wide expression censuses.

The Regulation of Sleep and Circadian Rhythms: The Role of Melatonin and Adenosine in Zebrafish

Sleep is a highly conserved behavioral state whose regulation is still unclear. In this thesis I initially briefly introduce the known sleep circuitry and regulation in vertebrates, and why zebrafish is seen as a good model to study sleep-regulation. I describe the existing two-process model of sleep regulation, which posits that the two processes C (circadian) and S (homeostatic) control timing of sleep-wake behavior. I then study the role melatonin plays in the circadian regulation of sleep using zebrafish. Firstly, we find that the absence of melatonin results in a reduction of sleep at night, establishing that endogenous melatonin is required for sleep at night. Secondly, melatonin mutants show a reduction in sleep in animals with no functional behavioral rhythms suggesting that melatonin does not require intact circadian rhythms for its effect on sleep. Thirdly, melatonin mutants do not exhibit any changes in circadian rhythms, suggesting that the circadian clock does not require melatonin for its function. Fourthly, we find that in the absence of melatonin, there is no rhythmic expression of sleep, suggesting that melatonin is the output molecule of process C. Lastly, we describe a connection between adenosine signaling (output molecules of process S), and melatonin. Following this we proceed to study the role adenosine signaling plays in sleep-wake behavior. We find that firstly, adenosine receptor A1 and A2 are involved in sleep- wake behavior in zebrafish, based on agonist/antagonist behavioral results. Secondly, we find that several brain regions such as PACAP cells in the rostral midbrain, GABAergic cells in the forebrain and hindbrain, Dopamine and serotonin cells in the caudal hypothalamus and sox2 cells lining the hindbrain ventricle are activated in response to the A1 antagonist and VMAT positive cells are activated in response to the A2A agonist, suggesting these areas are involved in adenosine signaling in zebrafish. Thirdly, we find that knocking out the zebrafish adenosine receptors has no effect on sleep architecture. Lastly, we find that while the A1 agonist phenotype requires the zfAdora1a receptor, the antagonist and the A2A agonist behavioral phenotypes are not mediated by the zfAdora1a, zfAdora1b and zfAdoraA2Aa, zfAdora2Ab receptors respectively.

Synthetic regulation of eukaryotic gene expression by noncoding RNA

Synthetic biological systems promise to combine the spectacular diversity of biological functionality with engineering principles to design new life to address many pressing needs. As these engineered systems advance in sophistication, there is ever-greater need for customizable, situation-specific expression of desired genes. However, existing gene control platforms are generally not modular, or do not display performance requirements required for robust phenotypic responses to input signals. This work expands the capabilities of eukaryotic gene control in two important directions.

For development of greater modularity, we extend the use of synthetic self-cleaving ribozyme switches to detect changes in input protein levels and convey that information into programmed gene expression in eukaryotic cells. We demonstrate both up- and down-regulation of levels of an output transgene by more than 4-fold in response to rising input protein levels, with maximal output gene expression approaching the highest levels observed in yeast. In vitro experiments demonstrate protein-dependent ribozyme activity modulation. We further demonstrate the platform in mammalian cells. Our switch devices do not depend on special input protein activity, and can be tailored to respond to any input protein to which a suitable RNA aptamer can be developed. This platform can potentially be employed to regulate the expression of any transgene or any endogenous gene by 3’ UTR replacement, allowing for more complex cell state-specific reprogramming.

We also address an important concern with ribozyme switches, and riboswitch performance in general, their dynamic range. While riboswitches have generally allowed for versatile and modular regulation, so far their dynamic ranges of output gene modulation have been modest, generally at most 10-fold. We address this shortcoming by developing a modular genetic amplifier for near-digital control of eukaryotic gene expression. We combine ribozyme switch-mediated regulation of a synthetic TF with TF-mediated regulation of an output gene. The amplifier platform allows for as much as 20-fold regulation of output gene expression in response to input signal, with maximal expression approaching the highest levels observed in yeast, yet being tunable to intermediate and lower expression levels. EC50 values are more than 4 times lower than in previously best-performing non-amplifier ribozyme switches. The system design retains the modular-input architecture of the ribozyme switch platform, and the near-digital dynamic ranges of TF-based gene control.

Together, these developments suggest great potential for the wide applicability of these platforms for better-performing eukaryotic gene regulation, and more sophisticated, customizable reprogramming of cellular activity.