Misregulation of Drosophila Myc Disrupts Circadian Behavior and Metabolism

Abstract

Drosophila Myc (dMyc) is highly conserved and functions as a transcription factor similar to mammalian Myc. We previously found that oncogenic Myc disrupts the molecular clock in cancer cells. Here, we demonstrate that misregulation of dMyc expression affects Drosophila circadian behavior. dMyc overexpression results in a high percentage of arrhythmic flies, concomitant with increases in the expression of clock genes cyc, tim, cry, and cwo. Conversely, flies with hypomorphic mutations in dMyc exhibit considerable arrhythmia, which can be rescued by loss of dMnt, a suppressor of dMyc activity. Metabolic profiling of fly heads revealed that loss of dMyc and its overexpression alter steady-state metabolite levels and have opposing effects on histidine, the histamine precursor, which is rescued in dMyc mutants by ablation of dMnt and could contribute to effects of dMyc on locomotor behavior. Our results demonstrate a role of dMyc in modulating Drosophila circadian clock, behavior, and metabolism.

Figure 1.. PER Immunoblot, Circadian Locomotor Rhythms, and PDF Expression of sLNvs in dMyc-Overexpressing Flies

(A) dMyc and PER protein levels determined by immunoblot in heads of tim-G > Myc+p35 flies compared to tim-G and + > Myc+p35 flies. Flies were entrained in light/dark 12:12-h cycle for 3–5 days. On the last day of entrainment, flies were snap frozen every 4–6 h and the heads were used to extract protein. α-Tubulin (α-Tub) is the loading control. Data are representative of three or more biological replicates. (B) Representative actogram of cry²⁴Pdf-G, cry²⁴Pdf-G > p35, cry²⁴Pdf-G > Myc+p35 rhythmic (RR), cry²⁴Pdf-G > Myc+p35 weakly rhythmic (WR), and cry²⁴Pdf-G > Myc+p35 arrhythmic (AR). Flies were entrained to a light/dark cycle for 3 days before being monitored in constant darkness over 8 days. (C and D) Decreased PDF expression in dorsal projections from sLNvs in dMyc-overexpressing flies with intact neuronal processes. Representative confocal image of brains from control flies (cry²⁴Pdf-G > p35) and dMyc-overexpressing flies (cry²⁴Pdf-G > Myc+p35) with membrane-labeled GFP (mCD8-GFP) (C) or without mCD8-GFP (D) subjected to immunofluorescence staining using antibodies directed against PDF (red in C; green in D) and PER (red in D) proteins at ZT1 and ZT13. A total of 12 brains from control flies and 34 brains from dMyc-overexpressing flies with mCD8-GFP were dissected, and the observations were consistent. Rectangles in (C) and arrows in (D): dorsal projections from sLNvs. Scale bars, 40 μm. See also Figures S1, S2, and S5, and Table 1.

Figure 2.. Hypomorphic dMyc Flies Exhibit Arrhythmic Behavior

(A) Schematic illustration (modified from Pierce et al., 2004) of the dm^P0 and dm⁴ alleles of the dMyc gene. (B) Picture of a dm^P0/Y fly versus its sibling control w/Y. C) Representative actogram of w/Y, rhythmic (RR) dm^P0/Y, and arrhythmic (AR) dm^P0/Y flies. Flies were entrained to a light/dark cycle for 3 days before being monitored in constant darkness over 7 days. (D and E) Period length (D) and rhythm strength (E) of rhythmic dm^P0/Y flies compared with control flies w/Y. (F) Picture of dm^P0/+, dm^P0/dm⁴, and dm^P0/dm⁴dMnt¹ flies. (G) Representative actogram of dm^P0/+, rhythmic (RR) dm^P0/dm⁴, arrhythmic (AR) dm^P0/ dm⁴, and dm^P0/ dm⁴dMnt¹. (H and I) Period length (H) and rhythm strength (I) of dm^P0/+, rhythmic dm^P0/dm⁴ (dm^P0/dm⁴), and dm^P0/ dm⁴dMnt¹ (dmP0/dm4dmnt) flies. Data are represented as mean ± SEM. *p < 0.05 and **p < 0.001 by Student’s t test. See also Figures S3 and S4, and Table 1.

Figure 3.. Metabolic Alteration in dMyc-Overexpressing and dMyc Hypomorphic Flies

(A) Relative enrichment of metabolites detected by gas chromatography-mass spectrometry (GC-MS) compared with Nor-leucine extracted from heads of control flies (cry²⁴Pdf-G > p35) or dMyc-overexpressing flies (cry²⁴Pdf-G > Myc+p35). Each data point is averaged from four pooled head extracts. Data are represented as mean ± SEM. *p < 0.05 and **p < 0.001 by Student’s t test. (B and C) Heatmap of metabolites (log value with normalized value between 0 and 1), detected by liquid chromatography-mass spectrometry (LC-MS) extracted from heads of dm^P0/+ (+), dm^P0/dm⁴ (dm⁴), and dm^P0/dm⁴dMnt¹ (dm⁴dMnt¹) flies at ZT3 (B) and ZT15 (C). (D) Model for the effect of dMyc and dMnt on the Drosophila molecular clock and locomotor activity. Schematic diagram of how dMyc and dMnt act on E-box-driven dMyc target genes (top). The bottom shows per/tim transcript oscillation (second column) and fly actogram (third column) in flies with dMyc overexpression (a), normal level of dMyc (b), low level of dMyc (c), and low level of dMyc with loss of dMnt (d). See also Figure S5.