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  • H3K4me3 Correlates with Transcription in Planarian Whole-Worm Tissue and Is Reduced at Distinct Loci upon RNAi of set1 versus mll1/2 (A) Schematic of ChIP-seq from dissociated whole-worm tissue; Drosophila S2 chromatin was 25% of total/ChIP. (B) Representative track of H3K4me3-ChIP DNA reads from wild-type whole worm tissue (wt-WW) aligned to the S. mediterranea genome. Red bars indicate MACS2-called peaks. (C) RNA-seq data from wt-WW for genes shown in (B). Error bars indicate SD across four biological replicates. (D) Meta-analysis of H3K4me3-ChIP data from wt-WW at all genes in the S. mediterranea genome with a MACS2-called peak; genes were binned according to the expression values indicated. (E) Histograms showing the distributions of the top 400 genes associated with H3K4me3 peak reductions in whole worms after set1(RNAi) (left) or mll1/2(RNAi) (right), each compared to control(RNAi); dashed line indicates −1.5 fold change (FC). (F) Venn diagram of genes associated with < −1.5 FC reductions in H3K4me3 upon set1(RNAi) (red circle) and mll1/2(RNAi) (green circle) in whole-worm tissue. (G) Representative tracks of H3K4me3-ChIP from RNAi-WW at gene loci with comparable H3K4me3 reductions (−2.0 FC). (H) Meta-analyses of H3K4me3 signal from set1(RNAi) or mll1/2(RNAi) WW-ChIP at Set1-affected loci (top plot) and MLL1/2-affected loci (bottom plot). Gray indicates standard error in meta-analyses (D and H). ChIP signal scale units are reads per million (B and G). ... Planarian Set1 and MLL1/2 Are Highly Conserved Proteins with Distinct RNAi-Knockdown Phenotypes (A) Schematic of the domain structure of the planarian proteins Set1 (SmedSet1) and MLL1/2 (SmedMLL1/2) in comparison to those of Drosophila (dSet1, dTrx) and human (hSet1, hMLL1, hMLL2). (B) Timeline detailing the RNAi feeding schedule used in all experiments, unless stated otherwise. (C and D) Live images of non-amputated control(RNAi), set1(RNAi) and mll1/2(RNAi) worms (C) and those at 7 days (left) and 17 days (right) post-amputation (D). Scoring of morphological phenotype is for a single representative experiment. ∗ indicates 3/8 fragments lysed before RD7. White arrowhead indicates early head regression. PR, photoreceptors. (E) In situ hybridization for the smedwi-1 stem cell marker in both set1(RNAi) and mll1/2(RNAi) non-amputated worms. (F) Confocal projections of the ciliated epithelium; cilia are labeled with antibody to acetylated-tubulin (green); nuclei are stained with DAPI (blue). Scale bars represent 500 μm (C and D), 200 μm (E), and 20 μm (F). See also Movies S1 and S2. ... Differences in H3K4me3 Correlate Significantly with Differences in the Transcriptional Profiles of Stem Cell and Differentiated Cell Populations (A) Schematic of ChIP-seq starting from FACS-isolated Smed cell populations; Drosophila S2 cells were >90% cells/ChIP. (B and C) Meta-analyses of H3K4me3-ChIP data from wild-type X1 stem cells (wt-X1) (B) and Xins differentiated cells (wt-Xins) (C) at all genes in the S. mediterranea genome with a MACS2-called peak in either population; genes are binned according to the expression values indicated. SE is in gray. (D) Representative tracks of H3K4me3-ChIP from wt-X1 and wt-Xins cells at gene loci with the indicated categories of H3K4me3 enrichment. Red bars indicate MACS2-called peaks, and purple bars indicate diffReps-called differential windows. ChIP signal scale units are reads per million. (E) Comparison of differential H3K4me3 and differential transcript expression at the gene loci in (D). (F) Venn diagram of all gene loci with enriched H3K4me3 (purple circle) and expression (blue circle) in X1 stem cells compared to Xins differentiated cells (pAdj < 0.01). The overlap (1,080) is significantly greater than the number expected by chance (317) (p = 3.5E-201, hypergeometric test).
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  • Dfd Is Expressed in SEG Motoneurons that Innervate Muscles Required for Head-Specific Motor Patterns (A) Diagram of a third-instar (L3) larval head highlighting the structures critical for mouth hook movements: mouth hook (MH), cephalopharyngeal skeleton (CPS), mouth hook elevator (MHE), mouth hook depressor (MHD), maxillary nerve (MN). The cibarial dilator muscle (CDM), used in this study as control muscle, is indicated. (B) MHE and MHD of DfdNAE667-Flp,tubP > GAL80 >, OK371::mCD8-GFP L3 larvae stained with Myosin to label muscles, DVGlut to mark functional synapses and GFP. (C) Diagram of a L3 CNS with the brain lobes (BLs), the ventral nerve cord (VNC), and the MN exiting the subesophageal ganglion (SEG) highlighted. (D) SEG from a DfdNAE667-Flp,tubP > GAL80 >, OK371::mCD8-GFP L3 larval CNS stained with Dfd, GFP, and DAPI for the DNA, arrowheads mark two to three SEG motoneurons that also express Dfd, and inset shows 3D reconstruction of these neurons. (E) Diagram of the head of a stage 16 Drosophila embryo with the MHE and MHD muscle precursors, the MN, and the SEG highlighted. (F and G) Close up of the SEG of stage 16 embryos expressing mCD8-GFP in Dfd-positive neurons by means of the Dfd-specific neuronal driver DfdNAE667-GAL4 (F) or the motoneuronal driver OK371-GAL4 (G). Arrowheads highlight Dfd-expressing neurons, which project their axons into the MN. Scale bars, 50 μm in (B) and (D), 20 μm in (F) and (G). See also Figure S1. ... Table S1. Neuronal and Mesodermal Dfd Target Genes Identified by ChIP-Seq Experiments Are Classified According to Their Gene Ontology Annotations... Drosophila
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