Browsing by Author "Littleton, Evan S."

Now showing 1 - 3 of 3
  • Thumbnail Image
    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.
  • Thumbnail Image
    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.
  • Thumbnail Image
    Preferential phosphatidylinositol 5-phosphate binding contributes to a destabilization of the VHS domain structure of Tom1
    Xiong, Wen; Tang, Tuo-Xian; Littleton, Evan S.; Karcini, Arba; Lazar, Iuliana M.; Capelluto, Daniel G. S. (Springer Nature, 2019-07-26)
    Tom1 transports endosomal ubiquitinated proteins that are targeted for degradation in the lysosomal pathway. Infection of eukaryotic cells by Shigella flexneri boosts oxygen consumption and promotes the synthesis of phosphatidylinositol-5-phosphate (PtdIns5P), which triggers Tom1 translocation to signaling endosomes. Removing Tom1 from its cargo trafficking function hinders protein degradation in the host and, simultaneously, enables bacterial survival. Tom1 preferentially binds PtdIns5P via its VHS domain, but the effects of a reducing environment as well as PtdIns5P on the domain structure and function are unknown. Thermal denaturation studies demonstrate that, under reducing conditions, the monomeric Tom1 VHS domain switches from a three-state to a two-state transition behavior. PtdIns5P reduced thermostability, interhelical contacts, and conformational compaction of Tom1 VHS, suggesting that the phosphoinositide destabilizes the protein domain. Destabilization of Tom1 VHS structure was also observed with other phospholipids. Isothermal calorimetry data analysis indicates that, unlike ubiquitin, Tom1 VHS endothermically binds to PtdIns5P through two noncooperative binding sites, with its acyl chains playing a relevant role in the interaction. Altogether, these findings provide mechanistic insights about the recognition of PtdIns5P by the VHS domain that may explain how Tom1, when in a different VHS domain conformational state, interacts with downstream effectors under S. flexneri infection.