Chromatin signatures at Notch-regulated enhancers reveal large-scale changes in H3K56ac upon activation.

The conserved Notch pathway functions in diverse developmental and disease-related processes, requiring mechanisms to ensure appropriate target selection and gene activation in each context. To investigate the influence of chromatin organisation and dynamics on the response to Notch signalling, we partitioned Drosophila chromatin using histone modifications and established the preferred chromatin conditions for binding of Su(H), the Notch pathway transcription factor. By manipulating activity of a co-operating factor, Lozenge/Runx, we showed that it can help facilitate these conditions. While many histone modifications were unchanged by Su(H) binding or Notch activation, we detected rapid changes in acetylation of H3K56 at Notch-regulated enhancers. This modification extended over large regions, required the histone acetyl-transferase CBP and was independent of transcription. Such rapid changes in H3K56 acetylation appear to be a conserved indicator of enhancer activation as they also occurred at the mammalian Notch-regulated Hey1 gene and at Drosophila ecdysone-regulated genes. This intriguing example of a core histone modification increasing over short timescales may therefore underpin changes in chromatin accessibility needed to promote transcription following signalling activation.This work was supported by a BBSRC project grant [BB/J008842/1] to SJB, BA and SR and by a MRC programme grant [G0800034] to SJB. JL is the recipient of a scholarship from the China Scholarship Council Cambridge.This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.15252/embj.20148992

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

A combination of computational and experimental approaches identifies DNA sequence constraints associated with target site binding specificity of the transcription factor CSL.

Regulation of transcription is fundamental to development and physiology, and occurs through binding of transcription factors to specific DNA sequences in the genome. CSL (CBF1/Suppressor of Hairless/LAG-1), a core component of the Notch signaling pathway, is one such transcription factor that acts in concert with co-activators or co-repressors to control the activity of associated target genes. One fundamental question is how CSL can recognize and select among different DNA sequences available in vivo and whether variations between selected sequences can influence its function. We have therefore investigated CSL-DNA recognition using computational approaches to analyze the energetics of CSL bound to different DNAs and tested the in silico predictions with in vitro and in vivo assays. Our results reveal novel aspects of CSL binding that may help explain the range of binding observed in vivo. In addition, using molecular dynamics simulations, we show that domain-domain correlations within CSL differ significantly depending on the DNA sequence bound, suggesting that different DNA sequences may directly influence CSL function. Taken together, our results, based on computational chemistry approaches, provide valuable insights into transcription factor-DNA binding, in this particular case increasing our understanding of CSL-DNA interactions and how these may impact on its transcriptional control.BBSRC [BB/J008842/1 to S.J.B., B.A., Dr Steve Russell.]; National Institutes of Health (NIH) [CA178974 to R.A.K.] and a Leukemia and Lymphoma Society Scholar Award (to R.A.K.); Unilever (to R.T. and R.G.). China Scholarship Council Cambridge (to J.L.); BBSRC Studentship (to R.A.F.); NIH training [5T32ES007250 to A.N.C.]. Funding for open access charge: University RCUK Open Access Fund.This is the final published version, originally published by Oxford Journals and available at http://nar.oxfordjournals.org/content/early/2014/08/11/nar.gku730.abstract

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

Prdm12 specifies V1 interneurons through cross-repressive interactions with Dbx1 and Nkx6 genes in Xenopus.

International audienceV1 interneurons are inhibitory neurons that play an essential role in vertebrate locomotion. The molecular mechanisms underlying their genesis remain, however, largely undefined. Here, we show that the transcription factor Prdm12 is selectively expressed in p1 progenitors of the hindbrain and spinal cord in the frog embryo, and that a similar restricted expression profile is observed in the nerve cord of other vertebrates as well as of the cephalochordate amphioxus. Using frog, chick and mice, we analyzed the regulation of Prdm12 and found that its expression in the caudal neural tube is dependent on retinoic acid and Pax6, and that it is restricted to p1 progenitors, due to the repressive action of Dbx1 and Nkx6-1/2 expressed in the adjacent p0 and p2 domains. Functional studies in the frog, including genome-wide identification of its targets by RNA-seq and ChIP-Seq, reveal that vertebrate Prdm12 proteins act as a general determinant of V1 cell fate, at least in part, by directly repressing Dbx1 and Nkx6 genes. This probably occurs by recruiting the methyltransferase G9a, an activity that is not displayed by the amphioxus Prdm12 protein. Together, these findings indicate that Prdm12 promotes V1 interneurons through cross-repressive interactions with Dbx1 and Nkx6 genes, and suggest that this function might have only been acquired after the split of the vertebrate and cephalochordate lineages