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. 2020 Mar 23;9:e52676. doi: 10.7554/eLife.52676

Figure 8. Pdm3 controls expression of the synaptic gene Msp300 to regulate sleep ontogeny.

(A) Scheme of RNA-Seq and subsequent modifier screen. (B) Volcano plot of RNA-Seq data highlighting significant changes in gene expression with pdm3 knockdown compared to controls in mid-pupal brains. Labeled genes have −log10(padj) >1.3 and absolute value of log2(Fold Change)>0.5. Yellow = increased expression, blue = decreased expression upon pdm3 knockdown (n = 4 biological replicates per genotype, 40 brains per replicate for RNA-Seq). (C) Modifier screen with co-expression of RNAi targeting upregulated genes from RNA-Seq alongside pdm3 RNAi (n ≥ 16 per genotype/age). (D) Overlap of hits from RNA-Seq and pdm3 ENCODE ChIP-Seq experiments. (E) Control-normalized peaks of PDM3 binding within the Msp300 gene. The strongest binding peak occurs upstream of the first exon in the RD, RL and RB transcript isoforms (shaded gray box). (F) Quantification of TH+ staining in the dFSB with Msp300 RNAi and pdm3 RNAi (n = 7, 10, 13 left to right). RNA-Seq statistical analysis is detailed in Materials and methods. ****p<0.0001, *p<0.05; ANOVA with Tukey’s test (F).

Figure 8.

Figure 8—figure supplement 1. Confirmation of PDM3 protein reduction at the mid-pupal developmental timepoint used for RNA-Seq experiments.

Figure 8—figure supplement 1.

Figure 8—figure supplement 2. Additional modifier screen data and further characterization of Msp300 phenotype.

Figure 8—figure supplement 2.

(A) Modifier screen with co-expression of RNAi targeting upregulated genes from RNA-Seq alongside pdm3 RNAi, plotted as total sleep time in young versus mature flies of each genotype (n ≥ 16 per genotype/age). Overlaid identity line represents young = mature total sleep time. (B) Total sleep time in Elav-pdm3 RNAi with co-expression of Msp300 RNAi (n = 32, 40, 14, 38, 14, 42, 26, 24 left to right). (C) Confirmation of PDM3 protein reduction with co-expression of Msp300 RNAi line with strongest effect on modifying sleep ontogeny (VDRC #105694). (D) Total sleep time with Elav >Msp300 RNAi only (n = 14, 27, 10, 24, 12, 24, 12, 24 left to right). ****p<0.0001, ***p<0.001, *p<0.05; multiple Student’s t tests with Holm-Sidak correction, alpha = 0.05 (B,D).
Figure 8—figure supplement 3. Molecular and cellular interactions between Msp300 and PDM3.

Figure 8—figure supplement 3.

(A) Visualization of reads from RNA-Seq in pdm3 RNAi (top, dark pink) versus controls (bottom, light pink), indicating a greater read pileup in pdm3 RNAi in the first exon common to Msp300 isoforms RD, RL and RB (gray box). (B) Msp300 knockdown in R93F07+ neurons in the setting of pan-neuronal pdm3 RNAi and controls (n = 90, 52, 31, 49, 56, 53 left to right). ****p<0.0001; multiple Student’s t tests with Holm-Sidak correction, alpha = 0.05.