005, nonparametric Mann-Whitney
test). A similar 2 hr temperature increase had no effect on control (Pdf-Gal4/+, UAS-TrpA1/+, and Pdf-Gal4 > UAS-mCD8GFP) fly lines ( Figures S2A and S2B). Similar to the role of mammalian Mef2 in activity-dependent neuronal plasticity ( Fiore et al., 2009, Flavell et al., 2006 and Flavell et al., 2008), activation of PDF cells with TrpA1 in a Mef2 RNAi knockdown strain induced defasciculation of the s-LNv dorsal termini (DI > 30%) in only ∼40% of brains, in contrast to ∼90% in wild-type brains (data not shown); the DI difference is statistically significant ( Figure 2B, p = 0.01, nonparametric Mann-Whitney test). This was not due to the extra UAS, as addition of a control UAS-mCherry element to a background fly line did not decrease axonal defasciculation in response to TrpA1 activation ( Figures S2C and S2D). The incomplete effect of the Mef2 knockdown probably reflects residual Mef2 activity and/or the PLX4032 ic50 very strong effect of TrpA1 on firing. An additional possibility is that Mef2-independent pathways also contribute to activity-induced axonal defasciculation. To gain further insight into the molecular XAV-939 mouse mechanisms that underlie Mef2 function in the circadian system, direct Mef2 target genes were identified with chromatin prepared from Drosophila adult heads. We analyzed the data with genome-wide tiling arrays (ChIP-Chip) and an antibody against isoform D of
Mef2 over ( Sandmann et al., 2006). The same antibody had been successfully used for identification of Mef2 targets in Drosophila embryos ( Junion et al., 2005 and Sandmann et al., 2006). We also addressed rhythmic binding of Mef2 to its genomic targets, i.e., the ChIP-Chip analysis was done on chromatin from fly heads collected at six different time points spanning the 24 hr light-dark cycle. Mef2 binds to a large number of sites in the Drosophila genome ( Table S1), and many of these were previously identified as Mef2 targets
genes in Drosophila embryos ( Sandmann et al., 2006); the overlap between the two gene lists is statistically significant (data not shown). Modified Fourier analysis ( Wijnen et al., 2005) of the six time points revealed rhythmic oscillations of Mef2 binding to a significant fraction of these loci. Maximal Mef2 binding was always in the latter half of the night and early morning, from approximately ZT17 to ZT2 ( Figure S3A). This temporal pattern of Mef2 chromatin cycling is in agreement with the gene expression data, which show an increase of Mef2 transcript levels in PDF neurons during the night ( Kula-Eversole et al., 2010; Figure 5B), as well as with the described oscillations of Mef2 protein levels in these cells, with maximal Mef2 nuclear accumulation at ZT22 ( Blanchard et al., 2010). We further validated Mef2 binding as well as cycling on several promoters by qRT-PCR analysis of three independent experimental repeats ( Figure S3B; Table S2).
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