Dynamic modes of Notch transcription hubs conferring memory and stochastic activation revealed by live imaging the co-activator Mastermind

DeHaro- Arbona etal. eLife 2023;12:RP92083. DOI: https://doi.org/10.7554/eLife.92083 1 of 22 ReseARcH ARtIcLe Dynamic modes of Notch transcription hubs conferring memory and stochastic activation revealed by live imaging the coactivatorMastermind F Javier DeHaro- Arbona etal. Figures and figure supplements Research article Chromosomes and Gene Expression | Developmental Biology DeHaro- Arbona etal. eLife 2023;12:RP92083. DOI: https://doi.org/10.7554/eLife.92083 2 of 22 Figure 1. Enrichment and dynamics of Mastermind at E(spl)- C. (A) Schematic overview of live imaging system used. Salivary glands from Drosophila larvae (left) have large nuclei with polytene chromosomes (centre, grey shading) in which E(spl)- C locus is detected as a band (centre, red) by live imaging when labelled using Int (orange)/ParB (red) system (right and Figure1—figure supplement 1A). Recruitment of activation complexes, Figure 1 continued on next page Research article Chromosomes and Gene Expression | Developmental Biology DeHaro- Arbona etal. eLife 2023;12:RP92083. DOI: https://doi.org/10.7554/eLife.92083 3 of 22 (CSL [green], NICD [purple], and Mastermind [magenta]) and of corepression complexes (CSL and Hairless [brown]) is measured by their colocalisation with E(spl)- C. (B) Live imaging of GFP::Mam and GFP::CSL as indicated in relation to E(spl)- C marked by Int- ParB (magenta) in nuclei from Notch OFF (1151Gal4; UAS- LacZ) and Notch ON (1151Gal4; UAS- N∆ECD) salivary glands. CSL and Mam are enriched at E(spl)- C in Notch ON but not Notch OFF cells. Average enrichment: each pixel represents average enrichment of all aligned images, centred on E(spl)- C locus (0, grey bar). Enrichment profile: mean enrichment, with SEM, plotted on xaxis relative to position, y axis, centred on E(spl)- C (0). Grey area indicates region used for max enrichment. Max enrichment: mean of 10 pixels centred on E(spl)- C (Figure1—figure supplement 1B) (Mam OFF, n = 32, Mam OFF, n = 30; CSL OFF, n = 45, CSL ON, n = 28; for pvalues, see Supplementary file 1—Table 4). Box encompasses range between 0.25 and 0.75 quantile, whiskers extend to furthest points not considered outliers, bar marks median, cross marks mean, and each dot is the value for one nucleus. Scale bars represent 5 µm. Genetic combinations for all figures are provided in Supplementary file 1—Table 3. (C, C’) Dynamics of CSL::GFP (green), GFP::Mam (magenta), and Hairless::GFP (brown) at E(spl)- C in Notch ON cells measured by fluorescence recovery after photobleaching (FRAP). Recovery of the indicated proteins was measured in a point bleached region- of- interest centred on E(spl)- C and normalised by using another region and efficiency of bleaching. Dots indicate 50% recovery. Legend summarises numbers of nuclei (n) and time to 50% recovery (t1/2). Error represents the standard error of the mean (SEM). (C’) FRAP analysis of CSL and Mam in cells depleted for Hairless (1151Gal4; UAS- Hairless RNAi) controls from (C) are included for comparison. (D) Trajectory density at E(spl)- C relative to whole nucleus from SPT of CSL::Halo (green) and Halo::Mam (pink) in Notch OFF and Notch ON cells. Both CSL and Mam are significantly enriched in Notch ON. Box plots as in (B). CSLOFF, n = 5, CSLOFF, n = 7; Mam- OFF, n = 5, Mam- ON, n = 13; for number of trajectories, see Figure1—figure supplement 1F and for pvalues see Supplementary file 1—Table 4. (E, E’) Survival curves depicting duration of trajectories in SPT of the indicated Halo fusion proteins in whole nuclei (E) and at E(spl)- C (E'), in Notch ON cells. Whole nuclei H2B::Halo trajectories are included for comparison in both graphs. For number of trajectories, see Figure1—figure supplement 1F. Error bars represent 95% confidence intervals, obtained from bootstrapping with 100 resampled datasets (Efron and Tibshirani, 1994). Figure 1 continued Research article Chromosomes and Gene Expression | Developmental Biology DeHaro- Arbona etal. eLife 2023;12:RP92083. DOI: https://doi.org/10.7554/eLife.92083 4 of 22 Figure 1—figure supplement 1. Image analysis and effect of Hairless depletion on CSL and Mam. (A) Scheme of the E(spl)- C in the Drosophila genome. Orange arrow indicates the insertion site of the IntB sequence that recruits ParB, E(spl)- mβ and E(spl)- m3 are shaded in magenta. (B) Schematic overview of image analysis. Images were rotated and a region of interest (ROI) was selected centred at the E(spl)- C. ROIs from images were averaged to produce an average enrichment image. 2D images extracted with the ROI were averaged in the ydimension to generate an intensity Figure 1—figure supplement 1 continued on next page Research article Chromosomes and Gene Expression | Developmental Biology DeHaro- Arbona etal. eLife 2023;12:RP92083. DOI: https://doi.org/10.7554/eLife.92083 5 of 22 profile enrichment plot, where the mean intensity and SEM were represented. For max values, the 10 middle values of the profile enrichment were averaged and plotted. Box encompasses range between 0.25 and 0.75 quantile, whiskers extend to furthest points not considered outliers, bar marks median, cross marks the mean, and each dot is the value for one nucleus. (C) Two colour images of Mam and CSL (labelled with GFP and mCherry, respectively) in Notch ON cells. Graph depicts correlation of the enrichment levels. (D) mRNA levels of Hairless in control (white RNAi) and Hairless RNAi conditions measured by RT- qPCR and normalised to RPL32. (E) Average enrichment, intensity profile and max values for Mam (n = 25, 27) and CSL (n = 37, 31) in Hairless RNAi (H Ri) in comparison to control, white RNAi (w Ri). For pvalues, see Supplementary file 1—Table 4. (F) Graph shows the number of trajectories for each molecule, all but histones are Notch ON and separated into nuclear and at E(spl)- C. Trajectories were segregated into bound and diffusive, as depicted by the cartoon. Percentages of each population for CSL and Mam were calculated per nuclei, error represents SEM (percentage CSL vs. Mam p=0.003, n = 7, 13 nuclei). Diffusion coefficients were calculated per nucleus as well. Diffusion coefficient bound CSL vs. bound Mam (0.27 ± 0.04 µm2/s, 0.25 ± 0.01 µm2/s, p=0.63), diffusion coefficient diffusive CSL vs. bound Mam (0.01 ± 0.001 µm2/s, 0.01 ± 0.001 µm2/s, p=0.34). Figure 1—figure supplement 1 continued Research article Chromosomes and Gene Expression | Developmental Biology DeHaro- Arbona etal. eLife 2023;12:RP92083. DOI: https://doi.org/10.7554/eLife.92083 6 of 22 Figure 2. Hublike properties of recruited complexes in Notch ON nuclei. (A) Representative images of GFP::CSL in Notch ON nuclei containing an ectopic array of 12 or 48 CSL binding sites. GFP::CSL is recruited to E(spl)- C (yellow) and the ectopic array (blue). Average enrichment, enrichment profile as in Figure1B (ectopic array 12, n = 45, ectopic array 48, n = 40; E(spl)- C 12, n = 45, E(spl)- C 48, n = 40). (B) Domain organisation of the indicated proteins is diagrammed above the prediction scores (Metapredict V2) of protein disorder for each. Regions tested as intrinsically disordered Figure 2 continued on next page Research article Chromosomes and Gene Expression | Developmental Biology DeHaro- Arbona etal. eLife 2023;12:RP92083. DOI: https://doi.org/10.7554/eLife.92083 7 of 22 regions (IDRs) are indicated. (C) Average enrichments and enrichment profiles of NICDPEST IDR (Notch OFF, n = 62, Notch ON, n = 67) and CSL- nt IDR (Wilcoxon rank sum test: Notch OFF, n = 8, Notch ON, n = 40) at E(spl)- C in Notch OFF and Notch ON cells. (D) Average enrichments and enrichment profiles (as in Figure1B) of GFP::Mam at E(spl)- C in nuclei expressing intact (Nact) and IDR deleted (Nact ΔIDR) Notch constructs (Nact, n = 58, Nact ΔIDR, n = 55). p- Values in Supplementary file 1—Table 4. (E) Diagram illustrates concentric ring analysis of singleparticle trajectories at increasing distances from E(spl)- C. Graphs plot proportions of bound (dark shading) and diffusive (light shading) particles of Halo::Mam or Halo::CSL within each ring. Dashed lines indicate nuclear proportions of each population as indicated. For the number of trajectories, see Figure1—figure supplement 1F. Figure 2 continued Research article Chromosomes and Gene Expression | Developmental Biology DeHaro- Arbona etal. eLife 2023;12:RP92083. DOI: https://doi.org/10.7554/eLife.92083 8 of 22 Figure 2—figure supplement 1. Notch activation complexes exhibit hublike properties. (A) Fixed Notch ON whole glands and nuclei of GFP::CSL and GFP::Mam (first channel), costained to detect E(spl)- C locus and DNA (Hoechst). Top row, whole gland, and lower row, individual example nuclei, with 25 µm and 5 µm scale bars, respectively. (A) An example of a nucleus expressing GFP::Mam[nIDR], imaged over time. Yellow arrows mark two Figure 2—figure supplement 1 continued on next page Research article Chromosomes and Gene Expression | Developmental Biology DeHaro- Arbona etal. eLife 2023;12:RP92083. DOI: https://doi.org/10.7554/eLife.92083 9 of 22 droplets that fuse (10 s frame). Scale bar indicates 5 µm. (B) Average, profile and max enrichment of IDR constructs: CSL[ctIDR], NICD[IDR], Med1[IDR], Mam, Mam[nIDR] in Notch OFF and Notch ON cells (upper and lower rows). For pvalues, see Supplementary file 1—Table 4. Figure 2—figure supplement 1 continued Research article Chromosomes and Gene Expression | Developmental Biology DeHaro- Arbona etal. eLife 2023;12:RP92083. DOI: https://doi.org/10.7554/eLife.92083 16 of 22 Figure 5. Effects of Notch withdrawal on hub composition imply memory state. (A) Temperature paradigm: withdrawal of Notch activity using the thermosensitive Gal4/Gal80ts system; after a 24 hr ON period, larvae were transferred to 18C (Gal4 inhibited) for the indicated periods (top) and enrichment of mCherry::CSL and GFP::Mam at E(spl)- C imaged. Average enrichment, enrichment profiles, and max enrichment quantified as in Figure1B. 24 hr ON, n = 28, 24 hr ON, 4 hr OFF, n = 21, 24 hr ON, 8 hr OFF, n = 19. p- Values are summarised in Supplementary file 1—Table 4. (B) Figure 5 continued on next page Research article Chromosomes and Gene Expression | Developmental Biology DeHaro- Arbona etal. eLife 2023;12:RP92083. DOI: https://doi.org/10.7554/eLife.92083 17 of 22 Accessibility of enhancers at E(spl) -mβ and E(spl) -m3, in Notch ON and after Notch withdrawal (8 hr) was probed using ATAC- qPCR, as in Figure4B. (C) Recruitment of CSL::GFP and expression of E(spl)-m3, detected by single molecule fluorescent in situ hybridisation (smFISH) in the conditions indicated. Robust recruitment of CSL::GFP at E(spl)- C persists after switching to nonepermissive temperature (Notch OFF) for 24 hr, representative image and graph (green) with intensity quantifications for multiple nuclei (24 hr ON, n = 28, +24 hr OFF, n = 21). For smFISH, representative images from cytoplasm (upper; 1.8 mm3) and nucleus (lower; centred on E(spl)- C, blue arrow) are shown; scale bars represent 5 µm. RNA puncta are absent after 24 hr Notch OFF. Graphs show the number of cytoplasmic RNA puncta (left) from n = 30, 16 regions and RNA fluorescence intensity at E(spl)- C in the conditions indicated from n = 10, 5 nuclei, with boxplots as described before (Figure1B). p- Values in Supplementary file 1—Table 4. (D) Optogenetic paradigm used for smFISH: Conditions used to switch ON and OFF Notch activity using OptiC Notch (Townson etal., 2023). (E, F) Expression of E(spl)-m3, detected by smFISH in the conditions indicated. (E) Representative images from the nucleus (centred on E(spl)- C, blue arrows). Scale bars represent 5 µm. (F) Graphs for fluorescence intensity at E(spl)- C in the conditions indicated from n = 38, 21, and 43 nuclei, with boxplots as described before (Figure1B). p- Values in Supplementary file 1—Table 4. (G) Optogenetic paradigm used for live imaging: conditions used to switch ON and OFF Notch activity using OptiC Notch (Townson etal., 2023). (H) Recruitment of CSL::GFP and Mam::Halo at E(spl)- C, measured following blue light activation in OptiC Notch expressing tissues for 2 hr (ON) and after 4 hr in dark (OFF). GFP::CSL remains enriched, Halo::Mam is depleted. (I) Recruitment of Halo::Mam in preactivated nuclei compared to naïve nuclei; temporal profile of Mam::Halo enrichment (right) and time taken to max enrichment (left). Error bars in the profile represents the SEM, values were normalised from 0 to 1 (similar to FRAP recovery, see Figure1 and ‘Materials and methods’), n indicates the number of nuclei from >3 salivary glands. Boxplot parameters as in Figure1B; naïve, n = 11, preactivated n = 9. Figure 5 continued Research article Chromosomes and Gene Expression | Developmental Biology DeHaro- Arbona etal. eLife 2023;12:RP92083. DOI: https://doi.org/10.7554/eLife.92083 18 of 22 Figure 5—figure supplement 1. Notch deactivation leads to loss of E(spl)-m3 transcription. (A) Cartoon depicts temperature paradigm, similar to Figure5A. Expression of E(spl)-m3 in the conditions indicated, detected by fluorescent in situ hybridisation (smFISH). Upper panels show representative cytoplasmic sections (1.8 mm3) and lower signal at the E(spl)- C. (n = 22, 30,30) for cytoplasmic 24 hr ON, 24 hr ON + 4 hr OFF and 24 hr ON + 8 hr OFF; n = 4, 4, 4 for nuclear 24 hr ON, 24 hr ON + 4 hr OFF and 24 hr ON + 8 hr OFF. p- Values provided in Supplementary file 1—Table 4. Research article Chromosomes and Gene Expression | Developmental Biology DeHaro- Arbona etal. eLife 2023;12:RP92083. DOI: https://doi.org/10.7554/eLife.92083 19 of 22 Figure 6. Recruitment of Pol II and Med1 in Notch ON is infrequent and augmented by ecdysone. (A) GFP::Mam enrichment at E(spl)- C in Notch OFF and Notch ON conditions as in Figure1, represented with a violin plot with mean marked by a cross and median by a bar. Right graph: proportions of nuclei with significant enrichment (obtained by performing a Gaussian fit on the fluorescence intensity Images, see Figure6—figure supplement 1). Over 90% nuclei have significant enrichment (Notch OFF, n = 32, Notch ON, n = 30). (B) Recruitment of GFP::Rbp3, a subunit of Pol II, at E(spl)- C in Notch OFF and Notch ON nuclei. Images: example where enrichment is detected. Graph: proportions of nuclei with significant enrichment as in (A) (Notch OFF, n = 37, Notch ON, n = 43). All scale bars represent 5 µm. (C) Recruitment of GFP::Med1 at E(spl)- C in Notch OFF and Notch ON nuclei. Images: example where enrichment is detected. Graph: proportions of nuclei with significant enrichment as in (A) (Notch OFF and ON, n = 38, Figure 6 continued on next page Research article Chromosomes and Gene Expression | Developmental Biology DeHaro- Arbona etal. eLife 2023;12:RP92083. DOI: https://doi.org/10.7554/eLife.92083 20 of 22 62). All scale bars represent 5 µm. (D) Transcriptional activity in Notch ON cells detected using live imaging of GFP::MCP recruited to MS2 loops in transcripts produced by E(spl)bHLHmβ (see ‘Materials and methods’). Images: representative examples of active (upper) and inactive (lower) nuclei. Graph: Proportion of active nuclei (Notch ON, n = 53). (E) Recruitment of GFP::Med1, mCherry::Rbp1, and Halo::CSL. Med1 and Rbp1 recruitments are correlated. Images: example of corecruitment of Med1, Rbp1, and CSL. Graph: correlation plot of max enrichment per nucleus at E(spl)- C, (marked by Halo::CSL recruitment), n = 48. (F) Pretreatment with ecdysone in Notch ON conditions increases proportions of nuclei with recruitment of GFP::Rbp3, GFP::Med1 and with active transcription foci (MS2/MCP intensity) compared to controls (EtOH). Proportions of nuclei with significant enrichment defined as in (A). Rbp3 ctrl, ecdysone: n = 39, 36. Med1 ctrl, ecdysone: n = 34, 34. MS2 ON ctrl, ON ecdysone, OFF ecdysone: n = 52, 42, 22. Figure 6 continued Research article Chromosomes and Gene Expression | Developmental Biology DeHaro- Arbona etal. eLife 2023;12:RP92083. DOI: https://doi.org/10.7554/eLife.92083 21 of 22 Figure 6—figure supplement 1. Population fitting reveals infrequent enrichment of Pol II and Med1 but is augmented by ecdysone treatment. (A–D, F–H) Gaussian fitting of Mam enrichment data from Figure1 and of Rbp3, Med1, and MS2 in the indicated conditions. Violin plots show the distribution of the data with dots depicting intensity or enrichment per nucleus, x average and bar median. μ indicates population average and σ the standard deviation. (E) Correlation of GFP::Med1 and Halo::CSL, mCherry::Rbp1 and Halo::CSL. Research article Chromosomes and Gene Expression | Developmental Biology DeHaro- Arbona etal. eLife 2023;12:RP92083. DOI: https://doi.org/10.7554/eLife.92083 22 of 22 Figure 7. Model illustrating different modes of Notch transcription hub. In the absence of Notch activity (Notch OFF), target genes are inactive, CSL is complexed with corepressors (e.g. Hairless). Following Notch activation (Notch ON), released Notch Intra Cellular Domain (NICD) (purple) generates a localised high concentration of transcription factors around target enhancer(s) referred to as a ‘hub’ (pink). Open hub: CSL (green) recruitment and accessible chromatin can occur in the absence of Mam. Engaged hub: presence of Mam (magenta) favours recruitment of additional factors, including Mediator CDK8 module (orange). Active hub: productive transcription, transition to this mode, with core Mediator (orange) and Pol II (pale blue) enrichment, is stochastic (dotted arrow). The probability can be enhanced by secondary signal, such as provided by ecdysone. Memory hub: CSL enrichment and chromatin accessibility remain after withdrawal of NICD.