Enrichment and dynamics of Mastermind at E(spl)-C.

(A) Schematic overview of live imaging system used. Drosophila salivary glands have large nuclei with polytene chromosomes that facilitate imaging. E(spl)-C locus is labelled using Int (orange)/ParB (red) system. Activation complexes, [CSL (green), NICD (purple) and Mastermind (magenta)] and co-repression complexes [CSL and Hairless (brown)] are depicted.

(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. 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 S1A) (Mam OFF, n=32, Mam OFF, n = 30; CSL OFF, n = 45, CSL ON, n = 28, for p-values see Table S3). 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.

(C) Dynamics of CSL::GFP (green), GFP::Mam (magenta) and Hairless::GFP (brown) at E(spl)-C in Notch-ON cells measured by FRAP. 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 p-values see Table S3.

(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 S1D. Error bars represent 95% confidence intervals, obtained from bootstrapping with 100 resampled datasets 96.

See also Supplementary Figure S1.

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 are diagrammed above the prediction scores (IUPRED) 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 T3

(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.

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; yRNAi, 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 S3.

(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 S3.

See also Supplementary Figure S3.

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

(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 were included in Notch-ON combinations, details are provided in Table S4. Scale bars represent 5um.

(B) Accessibility of Notch regulated enhancers adjacent to E(spl)bHLH-mβ and E(spl)bHLH-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 S3

(D-D’) Expression of E(spl)bHLH-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 non-permissive temperatures 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 S3.

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

(C) Expression of E(spl)bHLH-m3, detected by smFISH in the conditions indicated; representative images from cytoplasm (upper; 1.8 mm3) and nucleus (lower; centred on E(spl)-C, blue arrow). Representative images are shown, scale bars represent 5um. Graphs show number of RNA puncta (cytoplasmic) from n = 22, 30 and 30 regions, with boxplots as described before (Figure 1B). p-values in Table S3.

(D) Optogenetic paradigm: Conditions used to switch ON and OFF Notch activity using OptiC Notch 67.

(E) Recruitment of CSL::GFP and Mam::Halo at E(spl)-C, measured following blue light activation for 2 hours (ON) and after 4 hours in dark (OFF). GFP::CSL remains enriched, Halo::Mam is depleted.

(F) 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::Rbp1, 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::Rbp1, 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” (orange). 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 (yellow). Active hub: productive transcription, transition to this mode, with core Mediator (yellow) 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.