Transcription and chromatin dynamics in the Notch signalling response

During normal development, different genes are expressed in different cell types, often directed by cell signalling pathways and the pre-existing chromatin environment. The highly-conserved Notch signalling pathway is involved in many cell fate decisions during development, activating different target genes in different contexts. Upon ligand binding, the Notch receptor itself is cleaved, allowing the intracellular domain to travel to the nucleus and activate gene expression with the transcription factor known as Suppressor of Hairless (Su(H)) in Drosophila melanogaster. It is remarkable how, with such simplicity, the pathway can have such diverse outcomes while retaining precision, speed and robustness in the transcriptional response. The primary goal of this PhD has been to gain a better understanding of this process of rapid transcriptional activation in the context of the chromatin environment. To learn about the dynamics of the Notch transcriptional response, a live imaging approach was used in Drosophila Kc167 cells to visualise the transcription of a Notch-responsive gene in real time. With this technique, it was found that Notch receptor cleavage and trafficking can take place within 15 minutes to activate target gene expression, but that a ligand-receptor interaction between neighbouring cells may take longer. These experiments provide new data about the dynamics of the Notch response which could not be obtained with static time-point experiments. The chromatin accessibility and nucleosome dynamics at Notch-responsive enhancers were also studied using a variety of molecular techniques. These experiments showed that enhancers occupied by Su(H) were highly accessible with a high level of nucleosome turnover, and that Notch signalling promoted a further increase in accessibility. The BRM complex, a SWI/SNF chromatin remodeller implicated in many cancers, was identified as essential for the high chromatin accessibility at these regions and the Notch response. This new insight into the link between a simple signalling pathway and chromatin remodelling could have implications for understanding the complicated process of development and what goes wrong in diseases like cancer

SWI/SNF chromatin remodeling controls Notch-responsive enhancer accessibility.

Notch signaling plays a key role in many cell fate decisions during development by directing different gene expression programs via the transcription factor CSL, known as Su(H) in Drosophila Which target genes are responsive to Notch signaling is influenced by the chromatin state of enhancers, yet how this is regulated is not fully known. Detecting a specific increase in the histone variant H3.3 in response to Notch signaling, we tested which chromatin remodelers or histone chaperones are required for the changes in enhancer accessibility to Su(H) binding. We show a crucial role for the Brahma SWI/SNF chromatin remodeling complex, including the actin-related BAP55 subunit, in conferring enhancer accessibility and enabling the transcriptional response to Notch activity. The Notch-responsive regions have high levels of nucleosome turnover which depend on the Brahma complex, increase in magnitude with Notch signaling, and primarily involve histone H3.3. Together these results highlight the importance of SWI/SNF-mediated nucleosome turnover in rendering enhancers responsive to Notch

Notch Mediates Inter-tissue Communication to Promote Tumorigenesis.

Disease progression in many tumor types involves the interaction of genetically abnormal cancer cells with normal stromal cells. Neoplastic transformation in a Drosophila genetic model of epidermal growth factor receptor (EGFR)-driven tumorigenesis similarly relies on the interaction between epithelial and mesenchymal cells, providing a simple system to investigate mechanisms used for the cross-talk. Using the Drosophila model, we show that the transformed epithelium hijacks the mesenchymal cells through Notch signaling, which prevents their differentiation and promotes proliferation. A key downstream target in the mesenchyme is Zfh1/ZEB. When Notch or zfh1 are depleted in the mesenchymal cells, tumor growth is compromised. The ligand Delta is highly upregulated in the epithelial cells where it is found on long cellular processes. By using a live transcription assay in cultured cells and by depleting actin-rich processes in the tumor epithelium, we provide evidence that signaling can be mediated by cytonemes from Delta-expressing cells. We, thus, propose that high Notch activity in the unmodified mesenchymal cells is driven by ligands produced by the cancerous epithelial. This long-range Notch signaling integrates the two tissues to promote tumorigenesis, by co-opting a normal regulatory mechanism that prevents the mesenchymal cells from differentiating

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

Neuronal ensembles sufficient for recovery sleep and the sedative actions of α2 adrenergic agonists

Do sedatives engage natural sleep pathways? It is usually assumed that anesthetic-induced sedation and loss of righting reflex (LORR) arise by influencing the same circuitry to lesser or greater extents. For the α2 adrenergic receptor agonist dexmedetomidine, we found that sedation and LORR were in fact distinct states, requiring different brain areas: the preoptic hypothalamic area and locus coeruleus (LC), respectively. Selective knockdown of α2A adrenergic receptors from the LC abolished dexmedetomidine-induced LORR, but not sedation. Instead, we found that dexmedetomidine-induced sedation resembled the deep recovery sleep that follows sleep deprivation. We used TetTag pharmacogenetics in mice to functionally mark neurons activated in the preoptic hypothalamus during dexmedetomidine-induced sedation or recovery sleep. The neuronal ensembles could then be selectively reactivated. In both cases, non-rapid eye movement sleep, with the accompanying drop in body temperature, was recapitulated. Thus, α2 adrenergic receptor-induced sedation and recovery sleep share hypothalamic circuitry sufficient for producing these behavioral states

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

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