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 S1A). Recruitment of activation complexes, [CSL (green), NICD (purple) and Mastermind (magenta)] and of co-repression complexes [CSL and Hairless (brown)] is measured by their colocalization 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-NΔECD) 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 x-axis 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 S1B) (Mam OFF, n=32, Mam OFF, n = 30; CSL OFF, n = 45, CSL ON, n = 28, For p-values see Table S4). 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 5um. Full genotypes for all figures are provided in Table S3.

(C, C’) Dynamics of CSL::GFP (green), GFP::Mam (magenta) and Hairless::GFP (brown) at E(spl)-C in Notch ON cells measured by 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 summarizes 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). CSL-OFF, n = 5, CSL-OFF, n = 7; Mam-OFF, n = 5, Mam-ON, n = 13; For number of trajectories see Figure S1F and for p-values see Table S4.

(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 S1F. Error bars represent 95% confidence intervals, obtained from bootstrapping with 100 resampled datasets 103.

Hub-like 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 IDRs are indicated.

(C) Average enrichments and enrichment profiles of NICD-PEST 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 Table S4

(E) Diagram illustrates concentric ring analysis of single particle 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 number of trajectories see Figure S1F.

See also Supplementary Figure S2.

Mam enrichment requires Mediator CDK module but is independent of transcription and CBP/p300.

(A-B) GFP::Mam recruitment levels and dynamics at E(spl)-C in Notch ON tissues treated with triptolide or DMSO as a control. (A) Average and Max enrichment as in Figure 1B, (DMSO n = 49, triptolide, n = 36). (B) FRAP recovery curves, plotted as in Figure 1C.

(C–F) Average enrichment and Max enrichment of GFP::Mam and GFP::CSL, as indicated, at E(spl)-C in Notch ON control and treated tissues. (C-D) No change in recruitment following inhibition (C, A485) or genetic knockdown (D, RNAi) of CBP/p300 compared to control, DMSO and yellow RNAi (yRi) respectively. (DMSO, n = 47, A485, n = 62; y RNAi, n = 26, CBP/p300 RNAi, n = 31). (E, F) Reduced recruitment of GFP::Mam (magenta) but not CSL::GFP (green) following (E) genetic knockdown of Med13 and (F) inhibition of CDK8 with Senexin B. (E: Mam control, n = 30, Mam Med13Ri, n = 45; CSL control, n=92, CSL Med13Ri, n = 36) and (F: Mam ctrl n = 27, Mam Senexin, n = 43; CSL ctrl, n = 34, CSL Senexin, n = 37).

(G) Genetic knockdown of Cdk8 results in decreased Mam recruitment compared to controls (ctrl, n=33, Cdk8 Ri, n = 32). Box plots in A-F as in Figure 1B. For p-values see Table S4.

(H) Average enrichment and max enrichment of Med13::YFP at E(spl)-C in Notch OFF, n = 28 and Notch ON, n = 31; For p-values see Table S4.

See also Supplementary Figure S3.

Mam is not necessary for CSL enrichment and chromatin opening but necessary for transcription and Mediator 13 enrichment.

(A-A’) Recruitment of GFP::Mam in Notch ON nuclei is perturbed by MamDN (1151-Gal4 UAS-MamDN) (A) while recruitment of GFP::CSL is unaffected (A’). Control UAS was included in Notch ON combinations, details of genotypes are provided in Table S3. Scale bars represent 5um.

(B-B’) Accessibility of Notch regulated enhancers adjacent to E(spl) -mβ and E(spl) -m3 under the conditions indicated was probed using ATAC-qPCR. Values were normalised to Rab11, error bars represent SEM. Closed control is a noncoding genomic region in chromosome 3 and open control’ Eip78, an ecdysone responsive region active in L3 larval stage.

(C) Average enrichment and max enrichment of Med13::YFP at E(spl)-C in Notch ON conditions only, and in combination with MamDN. Quantifications as in Figure 1B, OFF, n=28, ON, n = 31. For p-values see Table S4

(D-D’) Expression of E(spl)-m3, detected by smFISH in the conditions indicated; representative images from cytoplasm (upper; 1.8mm3) and nucleus (lower; centred on E(spl)-C, blue arrows). Scale bars represent 5um. Graphs (D’), number of RNA puncta (cytoplasmic, n = 18, 20 and 22) and locus intensity (nucleus, n = 6, 7 and 6) in Notch OFF, Notch ON ctrl and Notch ON MamDN as indicated, boxplots as described in Figure 1B.

See also Supplementary Figure S4.

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 24h 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 hrs ON, n = 28, 24 hrs ON, 4 hrs OFF, n = 21, 24 hrs ON, 8 hrs OFF, n = 19. p-values are summarized in Table S4.

(B) Accessibility of enhancers at E(spl) -mβ and E(spl) -m3, in Notch ON and after Notch withdrawal (8 hours) was probed using ATAC-qPCR, as in Figure 4B.

(C) Recruitment of CSL::GFP and expression of E(spl)-m3, detected by smFISH in the conditions indicated. Robust recruitment of CSL::GFP at E(spl)-C persists after switching to none-permissive temperature (Notch OFF) for 24hours, representative image and graph (green) with intensity quantifications for multiple nuclei (24 hrs ON, n = 28, + 24 hrs 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 5um. RNA puncta are absent after 24 hours Notch OFF. Graphs show 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 Table S4.

(D) Optogenetic paradigm used for smFISH: Conditions used to switch ON and OFF Notch activity using

OptiC Notch 69

(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 5um. (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 Table S4.

(G) Optogenetic paradigm used for live imaging: Conditions used to switch ON and OFF Notch activity using OptiC Notch 69.

(H) Recruitment of CSL::GFP and Mam::Halo at E(spl)-C, measured following blue light activation in OptiC Notch expressing tissues for 2 hours (ON) and after 4 hours 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 methods), n indicates the number of nuclei from >3 salivary glands. Boxplots parameters as in Figure 1B; naïve, n = 11, preactivated n = 9.

Recruitment of Pol II and Med1 in Notch ON is infrequent and is 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 Supplementary Figure S5). 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 5um.

(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, 62). All scale bars represent 5um.

(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 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 co-recruitment 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.

See also Supplementary Figure S5.

Model illustrating different modes of Notch transcription-hub.

In the absence of Notch activity (Notch OFF) target genes are inactive, CSL is complexed with co-repressors (e.g. Hairless). Following Notch activation (Notch ON), released NICD (purple) generates a localized 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.