Figure 5. Not regulates EC-gene expression and is required for chromatin accessibility.

(A) Venn diagram comparing EC-related genes (Blue; Korzelius et al., 2014), genes exhibiting reduced expression upon loss of Not in ECs (Green), and chromatin regions with reduced accessibility upon loss of Not in ECs identified by ATAC-seq (Orange). (B) Metascape analysis of shared Non-stop-down-regulated genes and Esg over-expression in ECs. (C) Venn diagram comparison of genes that exhibit reduced expression upon loss of either Non-stop or Hey in ECs, as well as genes in the vicinity of regions showing reduced accessibility upon loss of Non-stop. (D) Venn diagram of genes that exhibit reduced expression upon loss of Non-stop or Hey and of genes with reduced expression upon over expression of LamDm0 in ECs (E) GO analysis of genes downregulated by loss of Non-stop in and Esg over-expression in ECs exhibiting reduced accessibility. Observed gene count; number of genes identified from this group in both ATAC-seq and RNA-seq (F) Genome-wide alignment and MEME analysis of regions with reduced accessibility in the vicinity of down-regulated genes upon loss of Non-stop in ECs. TSS, transcriptional start site; TES, transcription end site. (G–H) Confocal images of the midgut tissue using α-Histone H1 (red), and expressing the indicated transgenes in ECs using the MyoIA-Gal4/Gal80ts system for forty-eight hours, DAPI marks DNA (blue). (G) UAS-LacZ (control) (H), UAS-Non-stop RNAi. Scale bar is 10 μM. (I, J) western-blot analysis of the indicated proteins derived from gut extract (I), or S2 Drosophila cell extract (J) Histone H3 and Actin serve as loading controls.

Figure 5—figure supplement 1. Analysis of Not-related RNA-seq and ATAC-seq.

(A) (A1) Metascape analysis of biological process in genes exhibiting reduce expression upon loss of non-stop in ECs and not over expression of progenitor TF Escargot in ECs. (A2) List of shared transcription factors that exhibited reduced expression either upon loss of non-stop in ECs and expression of progenitor TF Escargot in ECs.(A3) List of transcription factors that exhibited reduced expression either upon loss of non-stop in ECs not affected by expression of the progenitor TF Escargot in ECs. (B) Venn diagram comparing genes exhibiting enriched expression in differentiated gut cells (Blue), genes exhibiting reduced expression upon loss of Not in ECs (Green), and chromatin regions with reduced accessibility upon loss of Not in ECs identified by ATAC-seq (Orange). Venn diagram comparison of genes that exhibit reduced expression upon loss of either Not or expression of LamDm0 ECs, and EC-expressed genes (C) Venn diagram comparison of genes that exhibit reduced expression or accessibility upon loss of Not, and genes with reduced expression upon over expression of LamDm0. (D) Venn diagram comparison of genes that exhibit upregulation in expression upon loss of either Not or Hey in ECs or over-expression of LamDm0 in ECs. (E). Three principle components analysis of RNA-seq.(F–H) Expression of LamC suppresses the ectopic expression of proliferating cell nuclear antigen (PNCA)) isopropyl β-d-1-thiogalactopyranoside (IPTG)) in EC that are no longer differentiated (PCC**). Confocal images of the midgut tissue using the indicated antibodies. (F–G) G-TRACE analysis; (F) Control ECs (expressing UAS-LacZ) do not express PCNA, and are both RFP⁽⁺⁾ GFP⁽⁺⁾. (G) PPC** are GFP⁽⁺⁾ and RPF^(-) (PPC **) and express PCNA (purple). Arrow points to cells shown in the insets (individual channels). (H) ECs where Non-stop was eliminated using the MyoIA-Gal4/Gal80ts system for forty-eight hours ectopically express PCNA (purple), but not in cells that co-express UAS-LamC. Example of two cells is shown; the purple and red arrows point to the cells shown in the inset; The left cell exhibits high level of LamC (red) and low level of PCNA (purple), and the right cell exhibit low level of LamC and high level of PCNA.

Figure 5—figure supplement 2. Analysis of changes in chromatin actability upon loss of Not.

(A) Whole genome changes in chromatin accessibility unveiled by ATAC seq divided to clusters by location along gene regions and GO ontology of each cluster. (B) MEME analysis of cluster -enrichment in DNA binding sequences associated with the indicated TFs. (C) List of all genes in the vicinity of regions that exhibit increased accessibility upon loss of Non -stop in ECs.