Regulation of olfactory associative memory by the circadian clock output signal Pigment-dispersing factor (PDF)

ABSTRACTDissociation between the output of the circadian clock and external environmental cues is a major cause of human cognitive dysfunction. While the effects of ablation of the molecular clock on memory have been studied in many systems, little has been done to test the role of specific clock circuit output signals. To address this gap, we examined the effects of mutation of Pigment-dispersing factor (Pdf) and its receptor, Pdfr on associative memory in male and female Drosophila. Loss of PDF signaling significantly decreases the ability to form associative memory. Appetitive short-term memory (STM), which in wildtype is time-of-day (TOD)-independent, is decreased across the day by mutation of Pdf or Pdfr, but more substantially in the morning than in the evening. This defect is due to PDFR expression in adult neurons outside the core clock circuit and the mushroom body Kenyon cells. The acquisition of a TOD difference in mutants implies the existence of multiple oscillators that act to normalize memory formation across the day for appetitive processes. Interestingly, aversive STM requires PDF but not PDFR, suggesting that there are valence-specific pathways downstream of PDF that regulate memory formation. These data argue that the circadian clock uses circuit-specific and molecularly diverse output pathways to enhance the ability of animals to optimize responses to changing conditions.SIGNIFICANCE STATEMENTFrom humans to invertebrates, cognitive processes are influenced by organisms’ internal circadian clocks, the pace of which is linked to the solar cycle. Disruption of this link is increasingly common (e.g. jetlag, social jetlag disorders) and causes cognitive impairments that are costly and long-lasting. A detailed understanding of how the internal clock regulates cognition is critical for the development of therapeutic methods. Here, we show for the first time that olfactory associative memory in Drosophila requires signaling by Pigment-dispersing factor (PDF), a neuromodulatory signaling peptide produced only by circadian clock circuit neurons. We also find a novel role for the clock circuit in stabilizing appetitive sucrose/odor memory across the day.

The Paralogous Krüppel-like Factors 9 and 13 Regulate the Mammalian Cellular Circadian Clock Output Gene Dbp

An intricate transcription-translation feedback loop (TTFL) governs cellular circadian rhythms in mammals. Here, we report that the zinc finger transcription factor Krüppel-like factor 9 (KLF9) is regulated by this TTFL, it associates in chromatin at the core circadian clock and clock-output genes, and it acts to modulate transcription of the clock-output gene Dbp. Our earlier genome-wide analysis of the mouse hippocampus-derived cell line HT22 showed that KLF9 associates in chromatin with Per1, Per3, Dbp, Tef, Bhlhe40, Bhlhe41, Nr1d1, and Nr1d2. Of the 3514 KLF9 peaks identified in HT22 cells, 1028 contain E-box sequences to which the transcriptional activators CLOCK and BMAL1 may bind, a frequency significantly greater than expected by chance. Klf9 mRNA showed circadian oscillation in synchronized HT22 cells, mouse hippocampus, and liver. At the clock-output gene Dbp, KLF9 exhibited circadian rhythmicity in its association in chromatin in HT22 cells and hippocampus. Forced expression of KLF9 in HT22 cells repressed basal Dbp transcription and strongly inhibited CLOCK+BMAL1-dependent transcriptional activation of a transfected Dbp reporter. Mutational analysis showed that this action of KLF9 depended on 2 intact KLF9-binding motifs within the Dbp locus that are in close proximity to E-boxes. Knockout of Klf9 or the paralogous gene Klf13 using CRISPR/Cas9 genome editing in HT22 cells had no effect on Dbp expression, but combined knockout of both genes strongly impaired circadian Dbp mRNA oscillation. Like KLF9, KLF13 also showed association in chromatin with clock- and clock-output genes, and forced expression of KLF13 inhibited the actions of CLOCK+BMAL1 on Dbp transcription. Our results suggest novel and partly overlapping roles for KLF9 and KLF13 in modulating cellular circadian clock output by a mechanism involving direct interaction with the core TTFL.

Molecular convergence of clock and photosensory pathways through PIF3–TOC1 interaction and co-occupancy of target promoters

A mechanism for integrating light perception and the endogenous circadian clock is central to a plant’s capacity to coordinate its growth and development with the prevailing daily light/dark cycles. Under short-day (SD) photocycles, hypocotyl elongation is maximal at dawn, being promoted by the collective activity of a quartet of transcription factors, called PIF1, PIF3, PIF4, and PIF5 (phytochrome-interacting factors). PIF protein abundance in SDs oscillates as a balance between synthesis and photoactivated-phytochrome–imposed degradation, with maximum levels accumulating at the end of the long night. Previous evidence shows that elongation under diurnal conditions (as well as in shade) is also subjected to circadian gating. However, the mechanism underlying these phenomena is incompletely understood. Here we show that the PIFs and the core clock component Timing of CAB expression 1 (TOC1) display coincident cobinding to the promoters of predawn-phased, growth-related genes under SD conditions. TOC1 interacts with the PIFs and represses their transcriptional activation activity, antagonizing PIF-induced growth. Given the dynamics of TOC1 abundance (displaying high postdusk levels that progressively decline during the long night), our data suggest that TOC1 functions to provide a direct output from the core clock that transiently constrains the growth-promoting activity of the accumulating PIFs early postdusk, thereby gating growth to predawn, when conditions for cell elongation are optimal. These findings unveil a previously unrecognized mechanism whereby a core circadian clock output signal converges immediately with the phytochrome photosensory pathway to coregulate directly the activity of the PIF transcription factors positioned at the apex of a transcriptional network that regulates a diversity of downstream morphogenic responses.

Neural and non-neural contributions to sexual dimorphism of mid-day sleep in Drosophila: A pilot study

Many of the characteristics associated with mammalian sleep are also observed in Drosophila, making the fruit-fly a powerful model organism for studying the genetics of this important process. Among these similarities is the presence of sexual dimorphic sleep patterns, which in flies, is manifested as increased mid-day sleep (‘siesta’) in males, compared to females. Here, we have used targeted miss-expression of the gene transformer (tra) and tra2 to either feminise or masculinise specific neural and non-neural tissues in the fly. Feminization of males using three different GAL4 drivers which are expressed in the mushroom bodies induced a female-like reduced siesta, while the masculinisation of females using these drivers triggered the male-like increased siesta. We also observed a similar reversal of sex-specific sleep by miss-expressing tra in the fat body, a key tissue in energy metabolism and hormone secretion. In addition, the daily expression levels of takeout, an important circadian clock output gene, were sexually dimorphic. Taken together, our experiments suggest that sleep-sexual dimorphism in Drosophila is driven by multiple neural and non-neural circuits, within and outside the brain.