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.

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Figures

Figure 1.

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.

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.

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.

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.

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.

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