Browsing by Author "Kojima, Shihoko"

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    Arrhythmogenic mechanisms of acute cardiac infection
    Padget, Rachel Lee (Virginia Tech, 2022-04-06)
    Cardiovascular disease is the leading cause of death world-wide, with 42% of sudden cardiac death in young adults caused by myocarditis. Viruses represent the main cause of myocarditis, with adenovirus being a leading pathogen. However, it is not understood how adenoviruses cause sudden cardiac arrest. Myocarditis is defined by two phases, acute and chronic. The acute phase involves viral-mediated remodeling of subcellular structures in the myocardium, which is thought to contribute to arrhythmogenesis. The chronic phase is immune response-mediated, where the host immune system causes damage that induces gross remodeling of the heart, which can result in cardiac arrest or heart failure. Electrical impulses of the heart are propagated by cardiomyocytes, via gap junctions, ion channels, and intracellular junctions, creating the healthy heartbeat. Cx43, the primary gap junction protein in the myocardium, not only propagates electrical signals, but also anti-viral molecules. Viral targeting of gap junction function leads to reduced anti-viral responses in neighboring cells. However, reduced cellular communication would dangerously alter cardiac conduction. Using a cardiotropic adenovirus, MAdV-3, we find that viral genomes are significantly enriched in the heart, with a decrease of gap junction and ion channel mRNA in infected hearts, however, their protein levels were unchanged. Phosphorylation of Cx43 at serine 368, known to reduce gap junction open probability, was increased in infected hearts. Ex vivo optical mapping illustrated decreased conduction velocity in the infected heart and patch clamping of isolated cardiomyocytes revealed prolonged action potential duration, along with decreased potassium current density during infection. Pairing mouse work with human induced pluripotent stem cell-derived cardiomyocytes, we found that human adenovirus type-5 infection increased pCx43-Ser368 and perturbation of intercellular coupling, as we observed with in vivo MAdV-3 infection. Allowing adenovirus infection to progress in vivo, we find myocardium remodeling and immune cell infiltration. Together, these data demonstrate the complexity of cardiac infection from viral-infection induced subcellular alterations in electrophysiology to immune-mediated cardiomyopathy of cardiac adenoviral infection. Our data describe virally induced mechanisms of arrhythmogenesis, which could lead to the development of new diagnostic tools and therapies, to help protect patients from arrhythmia following infection.
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    Changes in poly(A) tail length dynamics from the loss of the circadian deadenylase Nocturnin
    Kojima, Shihoko; Gendreau, Kerry L.; Sher-Chen, Elaine L.; Peng, Gao; Green, Carla B. (Nature, 2015-11-20)
    mRNA poly(A) tails are important for mRNA stability and translation, and enzymes that regulate the poly(A) tail length significantly impact protein profiles. There are eleven putative deadenylases in mammals, and it is thought that each targets specific transcripts, although this has not been clearly demonstrated. Nocturnin (NOC) is a unique deadenylase with robustly rhythmic expression and loss of Noc in mice (Noc KO) results in resistance to diet-induced obesity. In an attempt to identify target transcripts of NOC, we performed “poly(A)denylome” analysis, a method that measures poly(A) tail length of transcripts in a global manner, and identified 213 transcripts that have extended poly(A) tails in Noc KO liver. These transcripts share unexpected characteristics: they are short in length, have long half-lives, are actively translated, and gene ontology analyses revealed that they are enriched in functions in ribosome and oxidative phosphorylation pathways. However, most of these transcripts do not exhibit rhythmicity in poly(A) tail length or steady-state mRNA level, despite Noc’s robust rhythmicity. Therefore, even though the poly(A) tail length dynamics seen between genotypes may not result from direct NOC deadenylase activity, these data suggest that NOC exerts strong effects on physiology through direct and indirect control of target mRNAs.
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    The Circadian Deadenylase Nocturnin Is Necessary for Stabilization of the iNOS mRNA in Mice
    Niu, Shuang; Shingle, Danielle L.; Garbarino-Pico, Eduardo; Kojima, Shihoko; Gilbert, Misty; Green, Carla B. (PLoS, 2011-11-02)
    Nocturnin is a member of the CCR4 deadenylase family, and its expression is under circadian control with peak levels at night. Because it can remove poly(A) tails from mRNAs, it is presumed to play a role in post-transcriptional control of circadian gene expression, but its target mRNAs are not known. Here we demonstrate that Nocturnin expression is acutely induced by the endotoxin lipopolysaccharide (LPS). Mouse embryo fibroblasts (MEFs) lacking Nocturnin exhibit normal patterns of acute induction of TNFα and iNOS mRNAs during the first three hours following LPS treatment, but by 24 hours, while TNFα mRNA levels are indistinguishable from WT cells, iNOS message is significantly reduced 20-fold. Accordingly, analysis of the stability of the mRNAs showed that loss of Nocturnin causes a significant decrease in the half-life of the iNOS mRNA (t1/2 = 3.3 hours in Nocturnin knockout MEFs vs. 12.4 hours in wild type MEFs), while having no effect on the TNFα message. Furthermore, mice lacking Nocturnin lose the normal nighttime peak of hepatic iNOS mRNA, and have improved survival following LPS injection. These data suggest that Nocturnin has a novel stabilizing activity that plays an important role in the circadian response to inflammatory signals.
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    Coordination of rhythmic RNA synthesis and degradation orchestrates 24-and 12-h RNA expression patterns in mouse fibroblasts
    Unruh, Benjamin A.; Weidemann, Douglas E.; Miao, Lin; Kojima, Shihoko (National Academy of Sciences, 2024)
    Circadian RNA expression is essential to ultimately regulate a plethora of downstream rhythmic biochemical, physiological, and behavioral processes. Both transcriptional and posttranscriptional mechanisms are considered important to drive rhythmic RNA expression; however, the extent to which each regulatory process contributes to the rhythmic RNA expression remains controversial. To systematically address this, we monitored RNA dynamics using metabolic RNA labeling technology during a circadian cycle in mouse fibroblasts. We find that rhythmic RNA synthesis is the primary contributor of 24-h RNA rhythms, while rhythmic degradation is more important for 12-h RNA rhythms. These rhythms were predominantly regulated by Bmal1 and/or the core clock mechanism, and the interplay between rhythmic synthesis and degradation has a significant impact in shaping rhythmic RNA expression patterns. Interestingly, core clock RNAs are regulated by multiple rhythmic processes and have the highest amplitude of synthesis and degradation, presumably critical to sustain robust rhythmicity of cell-autonomous circadian rhythms. Our study yields invaluable insights into the temporal dynamics of both 24-and 12-h RNA rhythms in mouse fibroblasts.
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    Critical role of deadenylation in regulating poly(A) rhythms and circadian gene expression
    Yao, Xiangyu; Kojima, Shihoko; Chen, Jing (2020-04)
    The mammalian circadian clock is deeply rooted in rhythmic regulation of gene expression. Rhythmic transcriptional control mediated by the circadian transcription factors is thought to be the main driver of mammalian circadian gene expression. However, mounting evidence has demonstrated the importance of rhythmic post-transcriptional controls, and it remains unclear how the transcriptional and post-transcriptional mechanisms collectively control rhythmic gene expression. In mouse liver, hundreds of genes were found to exhibit rhythmicity in poly(A) tail length, and the poly(A) rhythms are strongly correlated with the protein expression rhythms. To understand the role of rhythmic poly(A) regulation in circadian gene expression, we constructed a parsimonious model that depicts rhythmic control imposed upon basic mRNA expression and poly(A) regulation processes, including transcription, deadenylation, polyadenylation, and degradation. The model results reveal the rhythmicity in deadenylation as the strongest contributor to the rhythmicity in poly(A) tail length and the rhythmicity in the abundance of the mRNA subpopulation with long poly(A) tails (a rough proxy for mRNA translatability). In line with this finding, the model further shows that the experimentally observed distinct peak phases in the expression of deadenylases, regardless of other rhythmic controls, can robustly cluster the rhythmic mRNAs by their peak phases in poly(A) tail length and abundance of the long-tailed subpopulation. This provides a potential mechanism to synchronize the phases of target gene expression regulated by the same deadenylases. Our findings highlight the critical role of rhythmic deadenylation in regulating poly(A) rhythms and circadian gene expression. Author summary The biological circadian clock aligns bodily functions to the day-and-night cycle and is important for maintaining health. The rhythms in various biological processes ultimately stem from rhythmic gene expression in each single cell. Because several proteins in the mammalian core clock machinery are transcription factors, studies of mammalian circadian gene expression have focused on rhythmic transcriptional control. However, many recent studies have suggested the importance of rhythmic post-transcriptional controls. Here we use mathematical modeling to investigate how transcriptional and post-transcriptional rhythms jointly control rhythmic gene expression. We particularly focus on rhythmic post-transcriptional regulation of the mRNA poly(A) tail, a nearly universal feature of mRNAs which controls mRNA stability and translation. Our model reveals that the rhythmicities in poly(A) tail length and mRNA translatability are most strongly affected by the rhythmicity in deadenylation, the process that shortens the poly(A) tail. Particularly, the phases of poly(A) tail length and mRNA translatability are dominated by the phase of deadenylation. In light of our findings, rhythmic control of deadenylation deserves greater future attention in the field of circadian gene expression.
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    Crosstalk Signaling Between Circadian Clock Components and Iron Metabolism
    Schiffhauer, Samuel Peter (Virginia Tech, 2017-04-25)
    Circadian rhythms are daily molecular oscillations within cells ranging from prokaryotes to humans. This rhythm is self sustaining, and receives external cues in order to synchronize an organism's behavior and physiology with the environment. Many metabolites utilized in metabolic processes seem to follow a pattern of circadian oscillation. Iron, an essential component in cellular processes such as respiration and DNA synthesis, is obtained almost exclusively through diet, yet little is known about how the clock governs iron metabolism. The regulation of iron within the cell is very tightly controlled, as iron is highly reactive in the generation of oxidative stress and the excretion of excess iron is very limited. There are limited findings indicating that there are molecular ties between the circadian clock and the regulation of iron metabolism. The first half of my dissertation focuses on the role of the circadian clock in modulating expression of iron metabolic components. We found that key components of iron import, in TFRC, and export, in SLC40A1, show altered expression in response to changes in the expression of clock transcription components. Furthermore, in circadian synchronized HepG2 hepatocytes TFRC and SLC40A1 showed rhythms in their mRNA expression, although expression of these genes was highly altered in conditions of high iron availability. We also examined IREB2, which expresses a master regulator of iron concentration in IRP2. IRP2 showed rhythms in phase with circadian component PER2, and IRP2's rhythmicity was lost under iron overload conditions. We observed that the ability of these three critical iron metabolic components to respond to sudden increases in available iron was mitigated in cells with clock impairment. Whole cistrome and transcriptome analysis was used to determine that rhythmicity in TFRC and SLC40A1 are not equal in their recruitment of circadian protein binding or in the stage of transcription in which circadian rhythms are generated. The cumulative effect of all of this regulation is that rhythmic variation in intracellular hepatic ferrous iron is clock controlled. The second half of my dissertation focuses on understanding how iron uptake influences clock resetting. Initially, iron was added to the cells in the form of ferrous sulfate, or chelated out of the cells using 2-2'-dipyridyl and clock gene expression was monitored. Altered rhythmicity of these components was seen at both the mRNA and protein level in cells with disrupted iron homeostasis. Then, we measured changes in period, phase, and amplitude of these rhythms, ultimately using a luciferase reporter cell line to demonstrate that even slight changes in cellular iron produce an effect on rhythmic period. We find that the circadian clock and iron metabolism pathway are intimately related, and that the intracellular iron concentration plays a role in circadian clock behavior. Overall, our research illustrates the importance of the circadian clock in liver metabolism and physiology. Improper iron metabolism due to genetic or dietary shortcomings is common in humans, and our work builds on the importance of chronotherapy in treatment of these conditions. Conversely, our research into the effect intracellular iron has on the clock contributes to the growing body of research into how circadian clocks, especially the peripheral clock of the liver, receive input from a range of metabolites in conjunction with signals from the master oscillator of the suprachiasmatic nucleus.
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    Functional Labeling of Individualized Post-Synaptic Neurons using Optogenetics and trans-Tango
    Castaneda, Allison Nicole (Virginia Tech, 2023-07-11)
    Neural circuitry, or how neurons connect across brain regions to form functional units, is the fundamental basis of all brain processing and behavior. There are several neural circuit analysis tools available across different model organisms, but currently the field lacks a comprehensive method that can 1) target post-synaptic neurons using a pre-synaptic driver line, 2) assess post-synaptic neuron morphology, and 3) test behavioral response of the post-synaptic neurons in an isolated manner. This work will present FLIPSOT, or Functional Labeling of Individualized Post-Synaptic Neurons using Optogenetics and trans-Tango, which is a method developed to fulfill all three of these conditions. FLIPSOT uses a pre-synaptic driver line to drive trans-Tango, triggering heat-shock-dependent expression of post-synaptic optogenetic receptors. When heat shocked for a suitable duration of time, optogenetic activation or inhibition is made possible in a randomized selection of post-synaptic cells, allowing testing and comparison of function. Finally, imaging of each brain confirms which neurons were targeted per animal, and analysis across trials can reveal which post-synaptic neurons are necessary and/or sufficient for the relevant behavior. FLIPSOT is then tested within Drosophila melanogaster to evaluate the necessity and sufficiency of post-synaptic neurons in the Drosophila Heating Cell circuit, which is a circuit that functions to drive warmth avoidance behavior. FLIPSOT presents a new combinatory tool for evaluation of behavioral necessity and sufficiency of post-synaptic cells. The tool can easily be utilized to test many different behaviors and circuits through modification of the pre-synaptic driver line. Lastly, the success of this tool within flies paves the way for possible future adaptation in other model organisms, including mammals.
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    Genome-wide correlation analysis to identify amplitude regulators of circadian transcriptome output
    Littleton, Evan S.; Childress, Madison L.; Gosting, Michaela L.; Jackson, Ayana N.; Kojima, Shihoko (Nature Research, 2020-12-14)
    Cell-autonomous circadian system, consisting of core clock genes, generates near 24-h rhythms and regulates the downstream rhythmic gene expression. While it has become clear that the percentage of rhythmic genes varies among mouse tissues, it remains unclear how this variation can be generated, particularly when the clock machinery is nearly identical in all tissues. In this study, we sought to characterize circadian transcriptome datasets that are publicly available and identify the critical component(s) involved in creating this variation. We found that the relative amplitude of 13 genes and the average level of 197 genes correlated with the percentage of cycling genes. Of those, the correlation of Rorc in both relative amplitude and the average level was one of the strongest. In addition, the level of Per2AS, a novel non-coding transcript that is expressed at the Period 2 locus, was also linearly correlated, although with a much lesser degree compared to Rorc. Overall, our study provides insight into how the variation in the percentage of clock-controlled genes can be generated in mouse tissues and suggests that Rorc and potentially Per2AS are involved in regulating the amplitude of circadian transcriptome output.
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    Identification and Characterization of Transcripts Regulated by Circadian Alternative Polyadenylation in Mouse Liver
    Gendreau, Kerry L.; Unruh, Benjamin A.; Zhou, Chuanli; Kojima, Shihoko (Genetics Society of America, 2018-11)
    Dynamic control of gene expression is a hallmark of the circadian system. In mouse liver, approximately 5-20% of RNAs are expressed rhythmically, and over 50% of mouse genes are rhythmically expressed in at least one tissue. Recent genome-wide analyses unveiled that, in addition to rhythmic transcription, various post-transcriptional mechanisms play crucial roles in driving rhythmic gene expression. Alternative polyadenylation (APA) is an emerging post-transcriptional mechanism that changes the 3-ends of transcripts by alternating poly(A) site usage. APA can thus result in changes in RNA processing, such as mRNA localization, stability, translation efficiency, and sometimes even in the localization of the encoded protein. It remains unclear, however, if and how APA is regulated by the circadian clock. To address this, we used an in silico approach and demonstrated in mouse liver that 57.4% of expressed genes undergo APA and each gene has 2.53 poly(A) sites on average. Among all expressed genes, 2.9% of genes alternate their poly(A) site usage with a circadian (i.e., approximately 24 hr) period. APA transcripts use distal sites with canonical poly(A) signals (PASs) more frequently; however, circadian APA transcripts exhibit less distinct usage preference between proximal and distal sites and use proximal sites more frequently. Circadian APA transcripts also harbor longer 3UTRs, making them more susceptible to post-transcriptional regulation. Overall, our study serves as a platform to ultimately understand the mechanisms of circadian APA regulation.
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    Insights on the Regulation of the PERIOD 2 Gene in the Cellular Response to DNA Damage
    Jiang, Liang (Virginia Tech, 2019-05-24)
    Circadian rhythm is a ~24-h mechanism that keeps our physiology and behavior in synchrony with environmental changes. PERIOD2 (PER2) is a core component of the circadian clock and a candidate tumor suppressor as its knockout expression results in a cancer-prone animal. p53 is an effector in the DNA damage response and regulates downstream effectors by trans-activation. Recent studies in our lab show that PER2 can bind to p53, and regulates the trans-activation function. This project studied the subcellular distribution of PER2 in response to DNA damage, and explored the role of p53 in the regulation of PER2 subcellular distribution. We found that PER2 accumulates in the nucleus in response to DNA damage, and such accumulation is independent of p53. In addition, we analyzed Single Nucleotide Polymorphisms (SNP) of PER2 in the 1000 Genome project to gain insight onto how missense mutations in PER2 lay at the interface of p53:PER2 binding. In a separate project, we also performed bioinformatics analysis on the iron related genes to discuss the circadian regulation of iron genes in the liver. These findings shed light on the regulation of PER2 under genotoxic stress, genetic variations of Per2 in normal human population, and expression of circadian genes under iron controlled diets.
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    Interlocking mechanisms regulating the circadian clock response to DNA damage
    Zou, Xianlin (Virginia Tech, 2021-06-15)
    Almost all organisms have an endogenously generated and self-sustained time-keeping system that oscillates with a periodicity of about 24 h, namely the circadian clock, that help them adapt to daily environmental changes. Mammalian circadian rhythms are generated and maintained by transcription-translation feedback loops (TTFLs) and include post-translational modifications to help fine-tune the oscillation. Circadian rhythms control a broad range of cellular signaling pathways including those mechanisms involved in cell division and DNA damage response (DDR). We have previously established that the core clock component PERIOD2 (PER2) binds to the tumor suppressor protein p53, a key regulatory checkpoint component that modulates cell cycle progression and the cellular response to genotoxic stress. PER2 binding to p53 modulates p53's stability, cellular localization, and transcriptional activity. As described in Chapter 2, we now identified PER2 as a previously uncharacterized substrate for the ubiquitin E3 ligase mouse double minute 2 homolog (MDM2), an oncoprotein and negative regulator of p53. Our findings showed that the association between PER2 and MDM2 is independent of the presence of p53. In addition, MDM2 targets PER2 for ubiquitylation and degradation in a phosphorylation-independent fashion. Lastly, our studies showed that MDM2 collaborates with β-transducin repeat-containing proteins (β-TrCPs), an E3 ligase that targets PER2 for ubiquitylation in a phosphorylation-dependent manner, to control PER2 degradation and thus the length of circadian period. Because the p53:MDM2 pathway plays a critical role in the cellular response to genotoxic stress, the project described in Chapter 3 is based on the hypothesis that DNA damage caused by radiation shifts the circadian clock phase via the p53:PER2:MDM2 complex. Firstly, we generated Trp53KO (Trp53 gene encodes mouse p53) cell lines in NIH 3T3 Per2:dLuc reporter cells expressing luciferase driven by the Per2 promoter. Phase-response curves (PRCs) for Trp53WT and Trp53KO reporter cells were obtained in response to ionizing radiation (IR) treatments. Results indicated that Trp53 knockout did not affect radiation-induced circadian phase shifts, whereas increased p53 levels induced by transient inhibitor treatments prevented phase shifts when IR was performed at the trough of PER2 abundance. Additional mechanisms were unveiled that kinases ATM (Ataxia Telangiectasia Mutated), ATR (ATM- and Rad3-related) and CHK2 (Checkpoint Kinase 2) regulate radiation-induced phase shifts. Lastly, we found that CLOCK (Circadian Locomotor Output Cycles Kaput) and CRY1 (CRYPTOCHROME 1) were phosphorylated in response to radiation. Taken together, these results indicate that radiation-induced clock phase shifts involve the activity of kinases ATM, ATR and CHK2, and the modification in CLOCK and CRY1. Chapter 4 is a review of current findings about the interaction between circadian rhythms and the cell division cycle regulation pathway. The article highlights a multidisciplinary approach that combines mathematical modeling and experimental data to reveal how p53:PER2:MDM2 acts as a node controlling timely cell cycle progression. In summary, our work provided evidence that MDM2 targets PER2 for ubiquitylation and degradation in a phosphorylation-independent manner, and this influences circadian oscillation. Furthermore, the exploration of p53:PER2:MDM2 association shed light on how radiation-induced DNA damage shifts clock phase. These findings expose a crosstalk mechanism that senses DNA damage and shifts the clock system.
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    Investigation of in-situ nanoimprinting of cell surface receptors: potential of a novel technique in biomarker research
    Ahmed, Sadia (Virginia Tech, 2019-01-22)
    Biomarkers are biological characteristics that can be observed or measured during disease conditions, and compared to the healthy state. Biomarkers have been used in medical history to study disease progression, to develop drugs, or to predict drug efficacy. However, in complex diseases such as in cancer, biomarkers vary tremendously among patients and disease stages. Cell surface receptors, proteins that are located at the cell surface and deliver external signals into the cell, are a significant group of easily-detectable biomarkers. Along with the detection of particular biomarkers related to a disease, extensive characterization of expression patterns is necessary to optimize their application. Therefore, we designed a technique to imprint or capture the expression pattern of these receptors on silver nanoparticles. We incorporated branched molecules that can simultaneously bind to the target receptors and the nanoparticle surface. To develop the technique, we used melanocortin receptor 1 (MC1R), a receptor present at high levels on the surface of melanoma cells, as a test system. We determined optimum binding of this molecule in an established melanoma cell line, WM-266-4. We also synthesized a labeled molecule that was used to estimate the number of MC1R proteins on these cells. These studies indicate that this might be a promising approach for developing sensitive and cost-effective tools to characterize cell surface receptors in studying complex diseases and cell mechanisms.
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    Modeling the interactions of sense and antisense Period transcripts in the mammalian circadian clock network
    Battogtokh, Dorjsuren; Kojima, Shihoko; Tyson, John J. (PLoS, 2018-02-15)
    In recent years, it has become increasingly apparent that antisense transcription plays an important role in the regulation of gene expression. The circadian clock is no exception: an antisense transcript of the mammalian core-clock gene PERIOD2 (PER2), which we shall refer to as Per2AS RNA, oscillates with a circadian period and a nearly 12 h phase shift from the peak expression of Per2 mRNA. In this paper, we ask whether Per2AS plays a regulatory role in the mammalian circadian clock by studying in silico the potential effects of interactions between Per2 and Per2AS RNAs on circadian rhythms. Based on the antiphasic expression pattern, we consider two hypotheses about how Per2 and Per2AS mutually interfere with each other's expression. In our pre-transcriptional model, the transcription of Per2AS RNA from the non-coding strand represses the transcription of Per2 mRNA from the coding strand and vice versa. In our post-transcriptional model, Per2 and Per2AS transcripts form a double-stranded RNA duplex, which is rapidly degraded. To study these two possible mechanisms, we have added terms describing our alternative hypotheses to a published mathematical model of the molecular regulatory network of the mammalian circadian clock. Our pre-transcriptional model predicts that transcriptional interference between Per2 and Per2AS can generate alternative modes of circadian oscillations, which we characterize in terms of the amplitude and phase of oscillation of core clock genes. In our post-transcriptional model, Per2/Per2AS duplex formation dampens the circadian rhythm. In a model that combines pre- and post-transcriptional controls, the period, amplitude and phase of circadian proteins exhibit non-monotonic dependencies on the rate of expression of Per2AS. All three models provide potential explanations of the observed antiphasic, circadian oscillations of Per2 and Per2AS RNAs. They make discordant predictions that can be tested experimentally in order to distinguish among these alternative hypotheses.
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    Mutations that alter Arabidopsis flavonoid metabolism affect the circadian clock
    Hildreth, Sherry B.; Littleton, Evan S.; Clark, Leor C.; Puller, Gabrielle C.; Kojima, Shihoko; Winkel, Brenda S. J. (Society for Experimental Biology, 2022-02-26)
    Flavonoids are a well-known class of specialized metabolites that play key roles in plant development, reproduction, and survival. Flavonoids are also of considerable interest from the perspective of human health, as both phytonutrients and pharmaceuticals. RNA sequencing analysis of an Arabidopsis null allele for chalcone synthase (CHS), which catalyzes the first step in flavonoid metabolism, has uncovered evidence that these compounds influence the expression of genes associated with the plant circadian clock. Analysis of promoter-luciferase constructs further showed that the transcriptional activity of CCA1 and TOC1, two key clock genes, is altered in CHS-deficient seedlings across the day/night cycle. Similar findings for a mutant line lacking flavonoid 3′-hydroxylase (F3′H) activity, and thus able to synthesize mono- but not dihydroxylated B-ring flavonoids, suggests that the latter are at least partially responsible; this was further supported by the ability of quercetin to enhance CCA1 promoter activity in wild-type and CHS-deficient seedlings. The effects of flavonoids on circadian function were also reflected in photosynthetic activity, with chlorophyll cycling abolished in CHS- and F3′H-deficient plants. Remarkably, the same phenotype was exhibited by plants with artificially high flavonoid levels, indicating that neither the antioxidant potential nor the light-screening properties of flavonoids contribute to optimal clock function, as has recently also been demonstrated in animal systems. Collectively, the current experiments point to a previously unknown connection between flavonoids and circadian cycling in plants and open the way to better understanding of the molecular basis of flavonoid action.
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    Natural antisense transcript of Period2, Per2AS, regulates the amplitude of the mouse circadian clock
    Mosig, Rebecca A.; Castaneda, Allison N.; Deslauriers, Jacob C.; Frazier, Landon P.; He, Kevin L.; Maghzian, Naseem; Pokharel, Aarati; Schrier, Camille T.; Zhu, Lily; Koike, Nobuya; Tyson, John J.; Green, Carla B.; Takahashi, Joseph S.; Kojima, Shihoko (Cold Spring Harbor Laboratory Press, 2021-05-20)
    In mammals, a set of core clock genes form transcription–translation feedback loops to generate circadian oscillations. In mammals, a set of core clock genes form transcription–translation feedback loops to generate circadian oscillations. We and others recently identified a novel transcript at the Period2 (Per2) locus that is transcribed from the antisense strand of Per2. This transcript, Per2AS, is expressed rhythmically and antiphasic to Per2 mRNA, leading to our hypothesis that Per2AS and Per2 mutually inhibit each other’s expression and form a double negative feedback loop. By perturbing the expression of Per2AS,we found that Per2AS transcription, but not transcript, represses Per2. However, Per2 does not repress Per2AS, as Per2 knockdown led to a decrease in the Per2AS level, indicating that Per2AS forms a single negative feedback loop with Per2 and maintains the level of Per2 within the oscillatory range. Per2AS also regulates the amplitude of the circadian clock, and this function cannot be solely explained through its interaction with Per2, as Per2 knockdown does not recapitulate the phenotypes of Per2AS perturbation. Overall, our data indicate that Per2AS is an important regulatory molecule in the mammalian circadian clock machinery. Our work also supports the idea that antisense transcripts of core clock genes constitute a common feature of circadian clocks, as they are found in other organisms.
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    Non-coding RNA genes lost in Prader-Willi Syndrome stabilize target RNAs
    Kocher, Matthew Afshin (Virginia Tech, 2021-05-27)
    Prader-Willi Syndrome (PWS) is a genetic disease that results in abnormal hormone levels, developmental delay, intellectual disability, hypogonadism, and excessive appetite. The disease is caused by a de novo genetic deletion in chromosome 15. While many of the deleted genes have been identified, there is little known about their molecular function. There is evidence that a cluster of non-coding RNA genes in the deleted region known as the SNORD116 genes may be the most critical genes deleted in Prader-Willi Syndrome. It is unknown what the SNORD116 genes do at the molecular level, but recent evidence suggests they regulate the expression of other genes involved in the neuroendocrine system. Specifically, the SNORD116 gene is implicated in regulation of NHLH2, a transcription factor gene which plays a key role in development, hormonal regulation, and body weight. In this study we identify phylogenetically conserved regions of SNORD116 and predict interactions with its potential downstream RNA targets. We show that mouse Snord116 post-transcriptionally increases Nhlh2 RNA levels dependent on its 3'UTR and protects it from degradation within 45 minutes of its transcription. Additionally, a single nucleotide variant within Nhlh2 at the predicted Snord116 interaction site may disrupt Snord116's protective effect. This is the first observation of a molecular mechanism for Snord116, identifying its role in RNA stability, and leads us closer to understanding Prader-Willi Syndrome and finding a possible treatment. However, Snord116 in vitro knockdown or paternally inherited in vivo deletion fail to detect differential expression of Nhlh2, likely due to missing the key timepoint of Snord116 regulatory effects on Nhlh2 RNA soon after its transcriptional stimulation, and dependent on leptin signals. Furthermore, the hypothalamic mRNA expression profile of PWS mouse models fed a nutraceutical dietary supplement of conjugated linoleic acid reveals minimal overall changes, while the effect of diet may be stronger than genotype and potentially changes gene expression of metabolic molecular pathways.
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    A proteomics study to investigate the role of the neural niche in the development of metastatic HER2+ breast cancer
    Ahuja, Shreya (Virginia Tech, 2022-06-13)
    Advanced stage tumors can acquire the ability to divide uncontrollably, invade the surrounding matrix, and circulate through the bloodstream or lymphatic system to distant organs in a process known as metastasis. The brain, which is shielded from the environment by the blood brain barrier, offers an immunocompetent lodging spot for the circulating cancer cells. Therefore, it is a "popular" destination for metastasized cancers which even surpass the incidents of primary brain tumors. It is hypothesized that the disseminated cancer cells engage with the host cells of the perivascular neural niche in a poorly understood crosstalk of molecular factors, that in turn augment the metastatic colonization of cancer cells. A better understanding of this crosstalk is indispensable to apprehending the complexity of the metastasis process, and to facilitating the discovery of biomarkers that predict metastatic potential and improve patient prognosis. The larger goal of this study was to adopt a mass spectrometry-based systems biology approach to investigate the molecular mechanisms and regulatory networks that underlie the complex phenomenon of breast cancer propagation at the brain metastatic site. To achieve this, the study was divided in three sub-projects designed around the following objectives, i.e., (a) to comprehensively characterize the protein landscape of the neural niche or the brain microenvironment comprised of astrocytes, microglia and endothelial cells, (b) to explore the immunological protein networks activated in microglia cells upon stimulation with anti-inflammatory cytokines released by tumor cells in the brain, and (c) to investigate the protein-level changes elicited in HER2+ breast cancer cells when grown under conditions that simulate the brain microenvironment in-vitro. Detailed characterization of the neural niche enabled us to propose molecular mechanisms that allow for the seeding and outgrowth of metastasized cancer cells in the brain. The study further provided novel insights into the signaling networks that regulate the immune functions of the microglia and their role during cancer development. Lastly, an in-depth investigation of breast cancer cells cultured in the presence of neural niche factors revealed potential novel mechanisms of cancer cell dormancy during metastasis. Altogether, large-scale proteomics data generated in this work will help clarify the mechanisms of metastatic cancer development, and will lay the groundwork for future studies that aim at the discovery of novel biomarkers and druggable targets for the treatment of brain metastatic cancers.
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    Timing without coding: How do long non-coding RNAs regulate circadian rhythms?
    Mosig, Rebecca A.; Kojima, Shihoko (Academic Press-Elsevier, 2022-06-01)
    Long non-coding RNAs (lncRNAs) are a new class of regulatory RNAs that play important roles in disease development and a variety of biological processes. Recent studies have underscored the importance of lncRNAs in the circadian clock system and demonstrated that lncRNAs regulate core clock genes and the core clock machinery in mammals. In this review, we provide an overview of our current understanding of how lncRNAs regulate the circadian clock without coding a protein. We also offer additional insights into the challenges in understanding the functions of lncRNAs and other unresolved questions in the field. We do not cover other regulatory ncRNAs even though they also play important roles; readers are highly encouraged to refer to other excellent reviews on this topic.
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    Understanding the dynamics of rhythmic gene expression in mammalian cells
    Unruh, Benjamin Alex (Virginia Tech, 2023-06-16)
    In mammals, circadian rhythms are driven by a cell-autonomous core-clock mechanism consisting of over a dozen core-clock genes forming transcription-translation feedback loops. The core-clock mechanism also drives the rhythmic expression of downstream genes called clock-controlled genes, which are thought to be important for driving rhythmic biochemical and physiological processes. Mathematical models predict that for a gene to be rhythmically expressed, synthesis, degradation, or a combination of the two must be rhythmic. The purpose of this project was to investigate the contribution of synthesis and degradation of RNA to rhythmic gene expression. To systematically understand the contribution of synthesis, degradation, and other RNA dynamics to rhythmic gene expression, I used metabolic labeling and a novel computational pipeline to analyze transcriptomic data in synchronized NIH3T3 cells. I identified 685 rhythmically expressed RNAs with a period of 24-hour in my dataset, of those 389 were rhythmically synthesized and 24 were rhythmically degraded. Low amplitude degradation rhythms were detected more broadly in the 685 rhythmically expressed RNAs, but these were not statistically significant. Although synthesis was the primary driver of rhythmic 24-hour RNA expression, core-clock gene RNAs were regulated by both synthesis and degradation, presumably to sustain high amplitude of rhythmic expression. I also identified rhythmic RNA expression with a period of 12 and 8 hours; interestingly, degradation primarily drove rhythmic expression of these RNAs. Overall this dissertation revealed RNA dynamics that drive rhythmic gene expression. This will provide insights into how diverse circadian clock mechanisms ultimately drive tissue-specific rhythmic gene expression.