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Biological significance of VIVID photocycle length and the control of gene expression by the Neurospora clock / by Arko Dasgupta

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Author	Dasgupta, Arko
Title	Biological significance of VIVID photocycle length and the control of gene expression by the Neurospora clock / by Arko Dasgupta
Date	2015
Abstract	Sensing light from the environment using photoreceptors is of great adaptive significance to eukaryotes. A prominent feature of the photochemistry of these receptors is the photocycle length, the time taken to decay from the initial signaling light state back to the receptive dark state. Photoreceptors have distinct photocycle lengths and this suggests evolutionary selection of the photochemistry for biological function. However, the biological significance of the photocycle length is not understood as most in vivo studies with photoreceptors utilize true null or blind photoreceptor mutants. Neurospora crassa provides a fantastic template for studying photobiology and the plethora of molecular and genetic resources available for this organism have enabled us to test the significance of photoreceptor photocycle length of the VIVID (VVD) protein. Using defined in vitro mutations that alter the photocycle length of the VVD protein over 4 orders of magnitude we have been able dissect the functional significance of the native photochemistry of the VVD protein. VVD interacts with the White Collar Complex (WCC), the primary transcription factor complex that drives light-responsive gene expression, and inhibits its transcriptional activity during a process known as photoadaptation. We find that a mutant VVD that shows extremely fast reversion from light state to dark state is unable to form a dynamic VVD-WCC pool of the size required for photoadaptation. Our study links in vitro photochemical studies with in vivo function and reveals previously unknown aspects of the photoadaptation machinery in Neurospora. Additionally, our work provides impetus to the photobiology field to study photoreceptor photocycle mutants in their native systems to truly appreciate the functional significance of the native photochemistry of photoreceptors. Neurospora is also a key model organism for studying circadian clocks and several major discoveries regarding the molecular basis of circadian clocks have been made in this fungus. Generally speaking circadian clocks are composed of three parts – the input, the core oscillator and the output. The core oscillator in Neurospora consists of a transcription-translation negative-feedback loop (TTFL). The Neurospora TTFL is comprised of a transcription factor complex, the WCC, which acts as the positive arm element of the TTFL and leads to the synthesis of the negative arm element of the TTFL, the FREQUENCY (FRQ) protein. FRQ in turn inhibits the transcriptional activity of the positive arm element in a time-delayed negative-feedback loop. Temporal changes in levels and activities of these two elements of the TTFL leads to the generation of sustained rhythmicity of the core circadian clock. The circadian clock controls rhythmic gene expression through cascades of transcription factor activation, and this involves WCC driven expression of target transcription factor genes as a pioneer step. Overall, the clock control of gene expression allows an organism to anticipate environmental conditions and better phase-specify its physiology, metabolism and behavior in a manner that makes biological sense. Using high-temporal resolution sampling over 48 hours we have identified that ~10% to 40% of the Neurospora transcriptome can be expressed rhythmically under circadian control. Through numerous gene ontology analyses we conclude that genes expressed under circadian control show an enrichment in pathways involving metabolism, protein synthesis and stress responses. Temporal segregation of peak gene expression of these genes reveals that catabolism, energy production, and precursor assembly are favored during daytime whereas biosynthesis of cellular components (and hence growth) is favored during nighttime. Additionally, using high-throughput promoter activity-reporter assays we find that the correlation between rhythmicity at the promoter level and rhythmicity at the mRNA level is poor, suggesting a role of post-transcriptional regulation in rhythmic gene expression. Furthermore, we have discovered that binding of WCC to its target genes occurs during a discrete phase of the circadian cycle, yet this binding is insufficient to set the phase of circadian expression from these genes. This suggests modulation of the expression of the WCC target genes requires additional factors. Temperature compensation (T.C.) is a core property of circadian rhythms, yet it is also the least understood in terms of mechanism. T.C. ensures that the clock maintains about the same period when exposed to a physiological range of temperatures. Casein kinase II, which has important functions in the core clock in Neurospora, had been previously identified as being important in T.C. Thus, we developed a high-throughput screen to identify other kinases and phosphatases that could potentially be involved in the property of T.C. in Neurospora, and show that most kinase and phosphatase mutants display robust T.C. We have also used historical T.C. mutants such as prd-3 and prd-2;prd-3 as templates for understanding the biochemistry of T.C. Our studies identify PRD-2 as another potential player in the post-transcriptional regulation of clock function. Additionally, we show that the FRQ turnover-rate, which was until recently considered to be the primary determinant of period length, cannot explain overcompensation defect in the prd-3 strain. Moreover, we show that T.C. defect in the prd-3 strain is a combination of the defects in the activities of both the positive and negative elements of the Neurospora TTFL.
Description	1 online resource (xiii, 289 leaves) : illustrations (mostly color)
Format	application/pdf
College	Geisel School of Medicine at Dartmouth -- Department of Genetics.
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D)--Dartmouth College.
Copyright	Made available by the author under a Creative Commons Attribution-NonCommercial License.; Made available by the Trustees of Dartmouth College under a Creative Commons Attribution-NonCommercial License. https://creativecommons.org/licenses/by-nc/4.0/
DOI	https://doi.org/10.1349/ddlp.1953

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Biological significance of VIVID photocycle length and the control of gene expression by the Neurospora clock / by Arko Dasgupta

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