Notch activation stimulates transient and selective binding of Su(H)/CSL to target enhancers - PubMed (original) (raw)
Notch activation stimulates transient and selective binding of Su(H)/CSL to target enhancers
Alena Krejcí et al. Genes Dev. 2007.
Abstract
The CSL [CBF1/Su(H)/Lag2] proteins [Su(H) in Drosophila] are implicated in repression and activation of Notch target loci. Prevailing models imply a static association of these DNA-binding transcription factors with their target enhancers. Our analysis of Su(H) binding and chromatin-associated features at 11 E(spl) Notch target genes before and after Notch revealed large differences in Su(H) occupancy at target loci that correlated with the presence of polymerase II and other marks of transcriptional activity. Unexpectedly, Su(H) occupancy was significantly and transiently increased following Notch activation, suggesting a more dynamic interaction with targets than hitherto proposed.
Figures
Figure 1.
EDTA elicits Notch activation in S2-N cells. (A) Diagram of E(spl) complex. Genes are indicated by arrows: basic helix–loop–helix genes (blue); Bearded-type (gray); not Notch responsive (white). (B) mRNA levels of E(spl) genes before and 30 min after EDTA treatment of S2-N cells. Bottom panel shows results plotted on a more sensitive scale. (C) m3 and m7 RNA levels in EDTA-treated S2-N cells ± γ-secretase inhibitor DFK-167 (300 μM). (D, top panel) Su(H) is detected as two bands in the total input and in α-Su(H) immunoprecipitates with and without EDTA treatment. (Bottom panel) Nicd is only present in the immunoprecipitated sample from EDTA-treated cells. Positions of molecular weight markers (in kilodaltons) are indicated for the bottom panel.
Figure 2.
Su(H) occupancy increases after Notch activation. ChIP with α-Su(H) (A,C) or α-Pol II (phosphorylated CTD) (B,D) antibodies in S2-N (A,B), and DmD8 (C,D) cells before (orange) or 30 min after (blue) EDTA. Precipitated DNA was quantified by real-time PCR. Each gene is represented by two or three fragments. (s) Su(H)-binding/enhancer region; (p) promoter; (o) ORF. A single 5′ region was amplified when Su(H) sites were close to the promoter. Control was rp49 ORF (rp). Results are average of three independent experiments (error bars indcate standard error of the mean). Increase in enrichment after activation of _m_β-s (P = 0.03) and _m3_-s (P = 0.04) in S2-N cells and of _m_δ-s (P = 0.04), _m_β-s (P = 0.04), _m3_-s (P = 0.003), _m6_-s (P = 0.002), and _m7_-s (P = 0.02) in DmD8 cells is significant.
Figure 3.
Time course of Su(H) occupancy and Nicd recruitment. (A) m3 mRNA levels in S2-N (blue) and S2 cells (orange) at the indicated times relative to EDTA treatment. (B–D) α-Su(H) (B,C) or α-Nicd (D) ChIP at the indicated times relative to EDTA treatment in S2-N (B) or DmD8 (C,D) cells. Enrichment of m3 enhancer (blue) and ORF (orange) (B) or m7 enhancer (blue) and rp49 ORF (control, gray) (C,D) were quantified. (E) Su(H) was immunoprecipitated at the indicated times and immunoprecipitates were probed to detect Su(H) (top panel, arrows) or Nicd (bottom panel, arrows). Approximate positions of molecular weight markers (in kilodaltons) are indicated. (F–H) Su(H) immunofluorescence (IF) (α-Su(H), red) (G,H) in S2-N cells treated as indicated. In F, distribution was quantified as described in the Supplemental Material. Differences between EDTA and control or mock cells were significant (P < 0.001) in S2-N cells. α-DMO (green) marks the nuclear envelope in G and H.
Figure 4.
Chromatin modifications across E(spl) genes before and after Notch activation in S2-N and DmD8 cells. ChIP was performed with the indicated antibodies in S2-N (A,B) and DmD8 (C,D) cells before (orange) and 25 min after (blue) EDTA and bound fragments were quantified by real-time PCR. Results are an average of three independent experiments (error bars indicate standard error of the mean). (B,D) For each column, levels were first calculated relative to input and then as a ratio to the equivalent sample from the α-H3 ChIP. Controls were rp49 (rp). In α-H3 ChIP, the decrease in enrichment of _m_β-o (P = 0.02) and _m3_-o (P = 0.05) in S2-N cells and of _m_δ-s (P = 0.006), _m_δ-p (P = 0.03), _m_β-s (P = 0.002), _m_α-p (P = 0.02), _m2_-p (P = 0.008), _m3_-s (P = 0.03), _m3_-p (P = 0.03), _m6_-s (P = 0.004), and _m7_-s (P = 0.03) in DmD8 cells are all significant.
Figure 5.
Dynamic model for Su(H) recruitment. Fast exchange and low residency of Su(H) (orange) when complexed with corepressors (turquoise). Following Notch activation, Su(H) complex with Nicd (dark purple) and Mam (light purple) is more stably associated with the DNA, possibly due to (1) interactions with other factors (gray, Pol II and associated factors) and/or (2) cooperative binding. This is accompanied by nucleosome (green) loss and histone modifications (Ac, me). Su(H) may form a complex with Nicd and Mam prior to DNA binding (a) or exchange could occur on transiently bound Su(H) (b). After recruitment, Nicd is subsequently modified and most likely degraded (Fryer et al. 2004), so Su(H) reverts to a lower occupancy state.
Similar articles
- Enhancers with cooperative Notch binding sites are more resistant to regulation by the Hairless co-repressor.
Kuang Y, Pyo A, Eafergan N, Cain B, Gutzwiller LM, Axelrod O, Gagliani EK, Weirauch MT, Kopan R, Kovall RA, Sprinzak D, Gebelein B. Kuang Y, et al. PLoS Genet. 2021 Sep 24;17(9):e1009039. doi: 10.1371/journal.pgen.1009039. eCollection 2021 Sep. PLoS Genet. 2021. PMID: 34559800 Free PMC article. - Structural and functional analysis of the repressor complex in the Notch signaling pathway of Drosophila melanogaster.
Maier D, Kurth P, Schulz A, Russell A, Yuan Z, Gruber K, Kovall RA, Preiss A. Maier D, et al. Mol Biol Cell. 2011 Sep;22(17):3242-52. doi: 10.1091/mbc.E11-05-0420. Epub 2011 Jul 7. Mol Biol Cell. 2011. PMID: 21737682 Free PMC article. - Molecular analysis of the notch repressor-complex in Drosophila: characterization of potential hairless binding sites on suppressor of hairless.
Kurth P, Preiss A, Kovall RA, Maier D. Kurth P, et al. PLoS One. 2011;6(11):e27986. doi: 10.1371/journal.pone.0027986. Epub 2011 Nov 18. PLoS One. 2011. PMID: 22125648 Free PMC article. - Notch-independent functions of CSL.
Johnson JE, Macdonald RJ. Johnson JE, et al. Curr Top Dev Biol. 2011;97:55-74. doi: 10.1016/B978-0-12-385975-4.00009-7. Curr Top Dev Biol. 2011. PMID: 22074602 Review. - Notch pathway: making sense of suppressor of hairless.
Bray S, Furriols M. Bray S, et al. Curr Biol. 2001 Mar 20;11(6):R217-21. doi: 10.1016/s0960-9822(01)00109-9. Curr Biol. 2001. PMID: 11301266 Review.
Cited by
- Advances in the role of membrane-bound transcription factors in carcinogenesis and therapy.
Deng J, Zhou J, Jiang B. Deng J, et al. Discov Oncol. 2024 Oct 15;15(1):559. doi: 10.1007/s12672-024-01414-1. Discov Oncol. 2024. PMID: 39404930 Review. - The cellular Notch1 protein promotes KSHV reactivation in an Rta-dependent manner.
DeCotiis-Mauro J, Han SM, Mello H, Goyeneche C, Marchesini-Tovar G, Jin L, Bellofatto V, Lukac DM. DeCotiis-Mauro J, et al. J Virol. 2024 Aug 20;98(8):e0078824. doi: 10.1128/jvi.00788-24. Epub 2024 Jul 8. J Virol. 2024. PMID: 38975769 - Novel Genome-Engineered H Alleles Differentially Affect Lateral Inhibition and Cell Dichotomy Processes during Bristle Organ Development.
Mönch TC, Smylla TK, Brändle F, Preiss A, Nagel AC. Mönch TC, et al. Genes (Basel). 2024 Apr 26;15(5):552. doi: 10.3390/genes15050552. Genes (Basel). 2024. PMID: 38790181 Free PMC article. - Dynamic modes of Notch transcription hubs conferring memory and stochastic activation revealed by live imaging the co-activator Mastermind.
DeHaro-Arbona FJ, Roussos C, Baloul S, Townson J, Gómez Lamarca MJ, Bray S. DeHaro-Arbona FJ, et al. Elife. 2024 May 10;12:RP92083. doi: 10.7554/eLife.92083. Elife. 2024. PMID: 38727722 Free PMC article. - Comprehensive genomic features indicative for Notch responsiveness.
Giaimo BD, Friedrich T, Ferrante F, Bartkuhn M, Borggrefe T. Giaimo BD, et al. Nucleic Acids Res. 2024 May 22;52(9):5179-5194. doi: 10.1093/nar/gkae292. Nucleic Acids Res. 2024. PMID: 38647081 Free PMC article.
References
- Agresti A., Scaffidi P., Riva A., Caiolfa V.R., Bianchi M.E., Scaffidi P., Riva A., Caiolfa V.R., Bianchi M.E., Riva A., Caiolfa V.R., Bianchi M.E., Caiolfa V.R., Bianchi M.E., Bianchi M.E. GR and HMGB1 interact only within chromatin and influence each other’s residence time. Mol. Cell. 2005;18:109–121. - PubMed
- Artavanis-Tsakonas S., Rand M.D., Lake R.J., Rand M.D., Lake R.J., Lake R.J. Notch signaling: Cell fate control and signal integration in development. Science. 1999;284:770–776. - PubMed
- Bailey A.M., Posakony J.W., Posakony J.W. Suppressor of Hairless directly activates transcription of Enhancer of split complex genes in response to Notch receptor activity. Genes & Dev. 1995;9:2609–2622. - PubMed
- Barolo S., Stone T., Bang A.G., Posakony J.W., Stone T., Bang A.G., Posakony J.W., Bang A.G., Posakony J.W., Posakony J.W. Default repression and Notch signaling: Hairless acts as an adaptor to recruit the corepressors Groucho and dCtBP to Suppressor of Hairless. Genes & Dev. 2002;16:1964–1976. - PMC - PubMed
- Bosisio D., Marazzi I., Agresti A., Shimizu N., Bianchi M.E., Natoli G., Marazzi I., Agresti A., Shimizu N., Bianchi M.E., Natoli G., Agresti A., Shimizu N., Bianchi M.E., Natoli G., Shimizu N., Bianchi M.E., Natoli G., Bianchi M.E., Natoli G., Natoli G. A hyper-dynamic equilibrium between promoter-bound and nucleoplasmic dimers controls NF-κB-dependent gene activity. EMBO J. 2006;25:798–810. - PMC - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Molecular Biology Databases