Sumoylation of Notch1 represses its target gene expression during cell stress

Abstract

The Notch signaling pathway is a key regulator of stem cells during development, and its deregulated activity is linked to developmental defects and cancer. Transcriptional activation of Notch target genes requires cleavage of the Notch receptor in response to ligand binding, production of the Notch intracellular domain (NICD1), NICD1 migration into the nucleus, and assembly of a transcriptional complex. Post-translational modifications of Notch regulate its trafficking, turnover, and transcriptional activity. Here, we show that NICD1 is modified by small ubiquitin-like modifier (SUMO) in a stress-inducible manner. Sumoylation occurs in the nucleus where NICD1 is sumoylated in the RBPJ-associated molecule (RAM) domain. Although stress and sumoylation enhance nuclear localization of NICD1, its transcriptional activity is attenuated. Molecular modeling indicates that sumoylation can occur within the DNA-bound ternary transcriptional complex, consisting of NICD1, the transcription factor Suppressor of Hairless (CSL), and the co-activator Mastermind-like (MAML) without its disruption. Mechanistically, sumoylation of NICD1 facilitates the recruitment of histone deacetylase 4 (HDAC4) to the Notch transcriptional complex to suppress Notch target gene expression. Stress-induced sumoylation decreases the NICD1-mediated induction of Notch target genes, which was abrogated by expressing a sumoylation-defected mutant in cells and in the developing central nervous system of the chick in vivo. Our findings of the stress-inducible sumoylation of NICD1 reveal a novel context-dependent regulatory mechanism of Notch target gene expression.

Fig. 1

Notch as a sumoylation target protein. a The sequence around the putative sumoylation site K1774/K1780/K1781/K1782 is located in the intracellular domain of Notch. b Sequence alignment of the Notch1 region surrounding the putative sumoylation site K1780 using the Clustal Omega web-based tool. The putative sumoylation site is highlighted in gray

Fig. 2

Notch1 is modified by SUMO. a GFP-Flag-NICD1 is modified by Histidine-SUMO1 (His-SUMO1) in HeLa cells. n = 4. b Activated Notch1 (ΔEN) is modified by transfected His-SUMO1 in HeLa cells. The samples were blotted with α-Val1744 recognizing NICD1. n = 4. c Heat shock increases the modification of Notch1 by SUMO1 and SUMO3. HeLa cells were transfected with GFP-Flag-NICD1 and His-SUMO1 (S1) or His-SUMO3 (S3). The samples were left untreated or heat-shocked for 1 h at 42 °C followed by a 1-h recovery at 37 °C (HS + recovery). n = 4. d Proteotoxic stress enhances Notch sumoylation. Transfected HeLa cells were left untreated, or were treated with the proteasome inhibitor Bortezomib at 37 °C for 8 or 16 h (h). n = 3. e Notch1 is modified by endogenous SUMO2/3. HeLa cells were heat-shocked and recovered as in (c). Hemagglutinin-SUMO2 (HA-SUMO2) was transfected as a positive control and pCMVKM empty vector (pCMV) as a negative control. n = 3. f Modification of Notch1 by endogenous SUMO1 is not detectable. The indicated samples were treated as in (e), but blotted with α-SUMO1. His-SUMO1 + GFP-Flag-NICD1 was transfected as a positive control. n = 3. g SENP1 and SENP2 deconjugate SUMO1 and SUMO2 from Notch1. COS7 cells were heat-shocked and recovered as in (c), lysed, and immunoblotted with α-GFP antibody. The sumoylated form of Notch1 is indicated with an arrow or a bracket and the unmodified form with an asterisk. n = 3

Fig. 3

SUMO enhances the nuclear localization of Notch1, interacts with Notch1 in the nucleus, and increases the levels of Notch1. a Sumoylation of Notch1 increases the nuclear localization of Notch1. COS7 cells were transfected with activated Notch1 (ΔEN) and/or His-SUMO1 (S1), or a control vector. Twenty-four hours after transfection the cells were left untreated or heat-shocked for 1 h at 42 °C followed by a 1-h recovery at 37 °C (HS). Images were taken with a Zeiss LSM510 META confocal microscope. n = 3. b Heat shock combined with transfected SUMO increases the nuclear localization of Notch1 most efficiently. Values are statistically significant at **p < 0.01 and represent averages from three independent experiments. Hundred cells in average were counted for each sample. c Notch1 is sumoylated in the nucleus. COS7 cells were heat-shocked and recovered as in (a). Nuclear fractions (NE) and cytoplasmic fractions (Cyt) were extracted and the samples were immunoblotted. GFP-Flag-NICD1 (NICD1) shows strong sumoylation in the nuclear fractions, whereas the cytoplasmic fractions show no conjugation of His-SUMO1 (S1) to NICD1. n = 3. d Consistent with (c), NICD1 cleaved from overexpressed ΔEN does not interact with His-SUMO1 (S1) in the cytoplasm, but only in the nucleus. The samples were treated as in (c), but immunoblotted with α-Notch1 C20. e COS7 cells were transfected as indicated, and  left untreated or treated with the protein translation inhibitor cycloheximide (CHX). The cells were heat-shocked at 42 °C followed by a 1-h recovery at 37 °C (HS + rec.), or  left untreated, and the samples were lysed and immunoblotted. f The levels of Notch1 are increased with increasing amounts of transfected SUMO1. COS7 cells were transfected with GFP-Flag-NICD (NICD) and different amounts of His-SUMO1 (S1), heat-shocked and recovered, lysed, and immunoprecipitated against Notch1 with protein G-sepharose beads and α-Notch1 C20, and analyzed with immunoblotting. The sumoylated form of Notch1 is indicated with an arrow or a bracket and the unmodified form with an asterisk. The lysates showed less difference in the levels of NICD, presumably due to decreased sumoylation of Notch1 in the presence of desumoylating isopeptidases. n = 3

Fig. 4

Three-dimensional model of sumoylated Notch1 transcription complex. CSL (light green), MAML1 (purple), DNA (orange), ANK repeat domain (cyan), and the N-terminal part (until E1777) of the RAM peptide (cyan) are from the crystal structure of the Notch1 transcription complex (PDB 3V79; 56). The C-terminal part of the RAM peptide (A1778-D1790) containing the sumoylated K1780 (shown as a representative for sumoylation with cyan spheres), K1781 and K1782 was modeled, along with SUMO (green) and the E2-conjugating enzyme Ubc9 (wheat), using the crystal structure of sumoylated Ran GTPase-activating protein 1 (PDB 3UIP; 80) as a template for modeling

Fig. 5

Sumoylation decreases Notch1 activity and represses the expression of Notch1 target genes. a K1774/1780/1781/1782R GFP-Flag-NICD1 (TSM) cannot be sumoylated as efficiently as wild-type GFP-Flag-NICD1 (WT). The sumoylated form of Notch1 is indicated with an arrow and the unmodified form with an asterisk. n = 3. b Wild-type NICD1 localizes more potently in the nucleus than TSM. COS7 cells were transfected with GFP-Flag-NICD1 (WT) or TSM, and some samples with His-SUMO1 (S1) as indicated. Images were taken with a Zeiss LSM510 META confocal microscope. c TSM increases the activity of the Notch1 signaling pathway. Notch1 activity was measured from HeLa cells by the 12×CSL luciferase reporter reflecting the activity of the Notch1 signaling pathway. The values shown are normalized luciferase units. n = 3. d–f Sumoylation (S1) decreases the expression of Notch (N1) target genes Hes1, Hey1, and Hey2 during heat shock. The relative expression of mRNA was analyzed by quantitative RT-PCR. n = 3. g–i Sumoylation (S1) decreases expression of Notch1 (WT) target genes Hes1, Hey1, and Hey1, whereas TSM increases the expression of Hes1, Hey1, and Hey2. The relative expression of mRNA was analyzed by quantitative RT-PCR. n = 3. j, k SUMO (S1) represses the expression of the indicated Notch target genes during proteotoxic stress in the form of Bortezomib (Brz). The relative expression of mRNA was analyzed by quantitative RT-PCR n = 3

Fig. 6

Sumoylation-resistant Notch1 mutant retains capacity to activate Hes5 after heat shock. a–d In embryos not exposed to heat shock (CONTROL) there is a reduction in TUJ1 immunoreactivity and an increase in Hes5 expression both when electroporated with NOTCH1-WT (a, b) and NOTCH1-TSM (c, d). n = 3. e–h In embryos exposed to heat shock for 1 h at 55 °C (HEAT SHOCK) there is a reduction in Hes5 levels in embryos electroporated with NOTCH1-WT (e, f), whereas NOTCH1-TSM electroporation induced high levels of the Hes5 transcript (g, h). n = 3

Fig. 7

HDAC4 decreases Notch1 activity in the presence of SUMO. a Sumoylated Notch1 binds to CSL and remains bound to DNA. n = 4. b Blocking the expression of HDAC4 by siRNA increases Notch1 activity. Notch1 activity was measured from HeLa cells by the 12×CSL luciferase reporter reflecting the activity of the Notch1 signaling pathway. The values shown are normalized luciferase units. The difference in Notch1 activity is greater in samples with overexpressed SUMO1 (S1) n = 3. c HDAC4 represses the activity of wild-type GFP-Flag-NICD1 (WT) more than it represses the activity of the K1774/1780/1781/1782R GFP-Flag-NICD1 (TSM). n = 3. d SUMO increases Notch1–HDAC4 interaction. Control samples without antibody during IP are indicated with an “M” referring to mock. n = 3. e Consistent with the results in (c), K1774/1780/1781/1782R GFP-Flag-NICD1 (NICD TSM) decreases NICD1–HDAC4 interaction compared to wildtype NICD1 (NICD WT). f Notch interacts with HDAC4 on the DNA. n = 3. g The levels of HDAC4 as related to Notch in the immunoprecipitates in (e) were quantified. Values indicate the average of three independent experiments. n = 3. h–j Inhibition of HDAC4 expression by siRNA increases, and HDAC4 overexpression decreases the expression of Notch1 (N1) target genes Hes1, Hey1, and Hey2 in the presence of SUMO1 (S1). The relative expression of mRNA was analyzed by quantitative RT-PCR. n = 3

Fig. 8

A model of the involvement of SUMO and HDAC4 in the repression of Notch1 target gene expression. In the absence of SUMO, NICD binds to CSL leading to the recruitment of MAML and consequent activation of specific Notch target genes. Recruitment of HDAC4 to the NICD/CSL/MAML transcriptional complex by sumoylated NICD promotes HDAC4-mediated transcriptional repression