Notch after cleavage.

The discovery that Notch activation involves a proteolytic cleavage to release the intracellular domain (NICD) revolutionized the field of Notch signaling. It resulted in a simple model whereby the cleaved NICD enters the nucleus and activates expression of genes by forming a DNA bound complex with CSL. However, is it really this simple? The realization that the outcome from activating Notch varies greatly from cell to cell raised many questions about what governs the target gene selections in different cell types. Insights have come from recent genome-wide studies, which highlight the importance of tissue-specific transcription factors and epigenetics. Co-factors also have been identified that participate in the regulation of enhancers. Finally, it is generally assumed that once cleaved, NICD goes on to do its job, but with a burgeoning number of post-translations, it may not be that simple

Rme-8 depletion perturbs Notch recycling and predisposes to pathogenic signaling.

Notch signaling is a major regulator of cell fate, proliferation, and differentiation. Like other signaling pathways, its activity is strongly influenced by intracellular trafficking. Besides contributing to signal activation and down-regulation, differential fluxes between trafficking routes can cause aberrant Notch pathway activation. Investigating the function of the retromer-associated DNAJ protein Rme-8 in vivo, we demonstrate a critical role in regulating Notch receptor recycling. In the absence of Rme-8, Notch accumulated in enlarged tubulated Rab4-positive endosomes, and as a consequence, signaling was compromised. Strikingly, when the retromer component Vps26 was depleted at the same time, Notch no longer accumulated and instead was ectopically activated. Likewise, depletion of ESCRT-0 components Hrs or Stam in combination with Rme-8 also led to high levels of ectopic Notch activity. Together, these results highlight the importance of Rme-8 in coordinating normal endocytic recycling route and reveal that its absence predisposes toward conditions in which pathological Notch signaling can occur.This work was funded by an MRC programme grant [G0800034] to SJB. LAS was the recipient of a BBSRC PhD studentship. ES and TK were funded by the DFG [Sachbeihilfe KL 1028/5-­1].This is the author accepted manuscript. The final version is available from Rockefeller University Press via http://dx.doi.org/10.1083/jcb.20141100

Role of co-repressor genomic landscapes in shaping the Notch response.

Repressors are frequently deployed to limit the transcriptional response to signalling pathways. For example, several co-repressors interact directly with the DNA-binding protein CSL and are proposed to keep target genes silenced in the absence of Notch activity. However, the scope of their contributions remains unclear. To investigate co-repressor activity in the context of this well defined signalling pathway, we have analysed the genome-wide binding profile of the best-characterized CSL co-repressor in Drosophila, Hairless, and of a second CSL interacting repressor, SMRTER. As predicted there was significant overlap between Hairless and its CSL DNA-binding partner, both in Kc cells and in wing discs, where they were predominantly found in chromatin with active enhancer marks. However, while the Hairless complex was widely present at some Notch regulated enhancers in the wing disc, no binding was detected at others, indicating that it is not essential for silencing per se. Further analysis of target enhancers confirmed differential requirements for Hairless. SMRTER binding significantly overlapped with Hairless, rather than complementing it, and many enhancers were apparently co-bound by both factors. Our analysis indicates that the actions of Hairless and SMRTER gate enhancers to Notch activity and to Ecdysone signalling respectively, to ensure that the appropriate levels and timing of target gene expression are achieved

Activation of the Notch Signaling Pathway In Vivo Elicits Changes in CSL Nuclear Dynamics.

A key feature of Notch signaling is that it directs immediate changes in transcription via the DNA-binding factor CSL, switching it from repression to activation. How Notch generates both a sensitive and accurate response-in the absence of any amplification step-remains to be elucidated. To address this question, we developed real-time analysis of CSL dynamics including single-molecule tracking in vivo. In Notch-OFF nuclei, a small proportion of CSL molecules transiently binds DNA, while in Notch-ON conditions CSL recruitment increases dramatically at target loci, where complexes have longer dwell times conferred by the Notch co-activator Mastermind. Surprisingly, recruitment of CSL-related corepressors also increases in Notch-ON conditions, revealing that Notch induces cooperative or "assisted" loading by promoting local increase in chromatin accessibility. Thus, in vivo Notch activity triggers changes in CSL dwell times and chromatin accessibility, which we propose confer sensitivity to small input changes and facilitate timely shut-down

OptIC-Notch reveals mechanism that regulates receptor interactions with CSL

Peer reviewed: TrueFunder: University of Cambridge; doi: http://dx.doi.org/10.13039/501100000735Active Notch signalling is elicited through receptor–ligand interactions that result in release of the Notch intracellular domain (NICD), which translocates into the nucleus. NICD activates transcription at target genes, forming a complex with the DNA-binding transcription factor CSL [CBF1/Su(H)/LAG-1] and co-activator Mastermind. However, CSL lacks its own nuclear localisation sequence, and it remains unclear where the tripartite complex is formed. To probe the mechanisms involved, we designed an optogenetic approach to control NICD release (OptIC-Notch) and monitored the subsequent complex formation and target gene activation. Strikingly, we observed that, when uncleaved, OptIC-Notch sequestered CSL in the cytoplasm. Hypothesising that exposure of a juxta membrane ΦWΦP motif is key to sequestration, we masked this motif with a second light-sensitive domain (OptIC-Notch{ω}), which was sufficient to prevent CSL sequestration. Furthermore, NICD produced by light-induced cleavage of OptIC-Notch or OptIC-Notch{ω} chaperoned CSL into the nucleus and induced target gene expression, showing efficient light-controlled activation. Our results demonstrate that exposure of the ΦWΦP motif leads to CSL recruitment and suggest this can occur in the cytoplasm prior to nuclear entry

OptIC Notch reveals mechanism that regulates receptor interactions with CSL.

Active Notch signalling is elicited through receptor-ligand interactions that result in release of the Notch intracellular domain (NICD), which translocates into the nucleus. NICD activates transcription at target genes forming a complex with the DNA-binding transcription factor CSL (CBF1/Su(H)/Lag-1) and co-activator Mastermind. Despite this, CSL lacks its own nuclear localisation sequence, and it remains unclear where the tripartite complex is formed. To probe mechanisms involved, we designed an optogenetic approach to control NICD release (OptIC-Notch) and monitored consequences on complex formation and target gene activation. Strikingly we observed that, when uncleaved, OptIC-Notch sequestered CSL in the cytoplasm. Hypothesising that exposure of a juxta membrane ΦWΦP motif is key to sequestration, we masked this motif with a second light sensitive domain in OptIC-Notch{ω}, which was sufficient to prevent CSL sequestration. Furthermore, NICD produced by light-induced cleavage of OptIC-Notch or OptIC-Notch{ω} chaperoned CSL into the nucleus and induced target gene expression, showing efficient light controlled activation. Our results demonstrate that exposure of the ΦWΦP motif leads to CSL recruitment and suggest this can occur in the cytoplasm prior to nuclear entry

Hairless binding is not detected at the <i>cut</i> or <i>wingless</i> Notch responsive genes.

<p><b>(A, B)</b> Profile of Su(H) and Hairless binding across genomic regions encompassing <i>wg</i> (A) and <i>cut</i> (B). Blue graphs: regions of Su(H) binding in control (Su(H) WT, fold enrichment, Log₂ scale -0.20 to 2.00;) and Notch over-expression conditions (Su(H) N[act] fold enrichment, Log₂ scale -0.20 to 2.50;<b>[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007096#pgen.1007096.ref037" target="_blank">37</a>]</b>). Brown graph: Hairless-GFP binding profile (fold enrichment, Log₂ scale -1.00 to 1.85). Grey graph: enrichment for H3K4me1 (scale: reads per million; <b>[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007096#pgen.1007096.ref064" target="_blank">64</a>]</b>). Gene models are depicted in blue, identified wing-disc enhancers indicated by cyan bar above, and regions significantly enriched for H3K27me3 <b>[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007096#pgen.1007096.ref064" target="_blank">64</a>]</b> indicated by grey bars below. (C, D) Removal of Hairless has limited effects on the expression of <i>wg</i> or <i>cut</i>. De-repression of <i>wg</i> (anti-Wg, purple, C; white C’) or <i>cut</i> (anti-Cut, purple, D; white D’) only occurs when cells with impaired Hairless (<i>H[P8]/H[P8]</i>, green, C,D) are close to the d/v boundary (arrows). No de-repression occurs at other locations (o). (E, F) Ectopic Notch activity elicits widespread activation of <i>wg</i> and <i>cut</i>. Ectopic expression of <i>wg</i> (anti-Wg, purple, E; white E’) occurs in clones of cells expressing NICD (green, E) at all locations. Expression of Cut (purple, F) and from a <i>cut</i> wing disc enhancer (<i>cut[2</i>.<i>1]-GFP</i>, green, F; white F’) occurs throughout the posterior compartment when NICD expression is driven by <i>en-Gal4</i>.</p

Hairless recruitment in Kc cells.

<p>(A) Venn diagram illustrating the proportion of Hairless bound regions in Kc cells that overlap with Su(H) binding. (B) Profile of Su(H) and Hairless across the <i>E(spl)</i> locus indicates co-binding. Graphs show GFP-Su(H) binding profile (blue graph: fold enrichment, Log₂ scale is -0.85 to 2.00), (1% FDR); Hairless-GFP binding profile (brown: fold enrichment, Log₂ scale is -0.90 to 3.74) and methylation enrichments from Hairless-Dam (orange: fold enrichment, Log₂ scale is -1.54 to 5.19). Gene models are depicted in blue. (C) Distribution of Hairless occupied regions in relation to chromatin states, shows strong preference for signature 3, “enhancer” state (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007096#sec010" target="_blank">methods</a> and <b>[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007096#pgen.1007096.ref034" target="_blank">34</a>]</b> for further details) (D) Knock-down of Hairless results in an increase in H3 acetylation similar to that seen with N activation (EGTA, 30 min). Graphs indicate differences in the enrichment profiles for H3K56ac ChIP from control and Notch activated (EGTA-treated) Kc cells or control and Hairless RNAi treated Kc cells, regions of significant difference are shaded (1% FDR; see <b>[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007096#pgen.1007096.ref034" target="_blank">34</a>]</b>).</p

Hairless recruitment in wing imaginal discs.

<p>(<b>A</b>) Venn diagram illustrating the proportion of Hairless bound regions that overlap with Su(H) binding in wild-type discs. (<b>B</b>) Profile of Su(H) and H across the <i>dpn</i> locus indicates co-binding. Blue graph: regions of Su(H) binding (fold enrichment, Log₂ scale -0.52 to 3.64; <b>[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007096#pgen.1007096.ref037" target="_blank">37</a>]</b>). Brown graph: Hairless -GFP binding profile (fold enrichment, Log₂ scale -1.00 to 1.85), horizontal lines below indicate regions of significant enrichment (peaks, 1% FDR). Grey graph: accessible chromatin identified by FAIRE <b>[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007096#pgen.1007096.ref063" target="_blank">63</a>]</b>. Gene models are depicted in blue, location of identified wing-disc enhancer in cyan <b>[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007096#pgen.1007096.ref038" target="_blank">38</a>]</b>. (<b>C</b>) Expression of <i>dpn</i> (purple) in wild-type wing disc, high levels of Dpn are detected at d/v boundary, lower levels in intervein regions. (D,D’) <i>dpn</i> (D, anti-Dpn, purple, D’, single channel white) is de-repressed in clones of cells with impaired Hairless (<i>H[P8]/H[P8]</i>, marked by GFP, green, D) at all locations in the wing disc.</p

SMRTER binding overlaps with Su(H) and Hairless.

<p>(A, B) Venn diagrams illustrating the proportion of SMRTER (SMR) bound regions in Kc cells that overlap with Su(H) binding (A) and with both Su(H) and Hairless (B). (C-E) Correlations between the most highly enriched bound regions (enrichment, Log₂ scale) for SMRTER and Su(H) (C), for Hairless and Su(H) (D) and for SMRTER and Hairless, (E). Only Hairless and Su(H) are very significantly correlated.</p