Figure 3: Identification of CREB/CRTC and histone acetylation target genes in the MBs. | Nature Communications

Figure 3: Identification of CREB/CRTC and histone acetylation target genes in the MBs.

From: Shifting transcriptional machinery is required for long-term memory maintenance and modification in Drosophila mushroom bodies

Figure 3

(a) Schematic diagram of the experimental design. (b) Subcellular localization of FLAG-KASH in the MBs. FLAG-KASH was expressed by MBp-LexA. (upper) anti-FLAG staining (green) and nuclear staining by TO-PRO-3 iodide were shown (magenta). Scale bars, 50 μm. (lower) Magnified view of the white squared area in the upper panel. Scale bars, 2 μm. The images are representative of three experimental replicates. (c) Nuclear staining of immunoprecipitated MB nuclei. Upper: the heads were homogenized and the nuclei were stained propidium iodide (magenta). Lower: the nuclei were immunoprecipitated using anti-FLAG antibody-conjugated beads. The nuclei were stained by propidium iodide (magenta). Scale bars, 25 μm. The images are representative of four experimental replicates. (d) The immunoprecipitated nuclei expressed FLAG-KASH. The precipitated nuclei in c were released from the beads and stained with anti-FLAG antibody (green). The nuclei were stained by propidium iodide (magenta). Scale bars, 25 μm. The images are representative of three experimental replicates. (e,h) ChIP-seq signals at the region near Bx. Purified MB nuclei expressing FLAG-KASH and prepared from naive flies or flies 1 day after spaced training were subjected to ChIP-seq analysis, using anti-H3K9Ac (n=3), anti-H4K16 (n=3) (e), anti-CREB (n=3) and anti-CRTC (n=3) antibodies (j). The sequencing data of input DNA was shown at the bottom. Increases in histone acetylation and CREB/CRTC-binding sites identified in this study are highlighted by light-yellow vertical bars. The y axes show the number of the mapped reads and the upper limits were adjusted to the number of the total reads in each samples. The base line indicates zero read. (f,g) Aggregate gene plot of H3K9Ac (f) and H4K16Ac (g). The mapped reads of naive samples or spaced-trained samples were pooled (n=3 for each, shown in green and orange lines, respectively) and were summarized with respect to the distance from the TSS and the transcriptional end site (TES). (i,j) Venn diagram showing overlaps of CREB/CRTC-binding genes where CRTC binding is increased and the genes with increases (i) or decreases (j) in H3K9Ac and H4K16Ac in 500 bp vicinity to TSS. The non-random overlaps indicated by hypergeomic test were shown by asterisks. NS, not significant; ***P<0.001.

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