p65-NF{kappa}B synergizes with Notch to activate transcription by triggering cytoplasmic translocation of the nuclear receptor corepressor N-CoR

Lluís Espinosa, Sara Santos, Julia Inglés-Esteve, Pura Muñoz-Canoves and Anna Bigas*

Centre Oncologia Molecular, Institut de Recerca Oncologica, Hospitalet, Barcelona 08907, Spain

* Author for correspondence (e-mail: abigas{at}iro.es )

Accepted 13 December 2001


    Summary
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Notch/RBP-J{kappa} and nuclear factor-{kappa}B (NF{kappa}B) complexes are key mediators of the progression of many cellular events through the activation of specific target gene transcription. Independent observations have shown that activation of Notch-dependent transcription generally correlates with inhibition of differentiation. In contrast, activated NF{kappa}B complexes are required for progression of differentiation in several systems. Although some interactions between both pathways have been observed, the physiological significance of their connection is unclear. We have now demonstrated that the increase in p65-NF{kappa}B protein levels enhances Notch-mediated activation of the Hes1 promoter up to three-fold. This effect does not require NF{kappa}B transcriptional activity, and it is independent of the previously described interaction between Notch and p50-NF{kappa}B. Furthermore, we show that p65-NF{kappa}B can modulate subcellular localization of the transcriptional corepressor N-CoR, abrogating N-CoR mediated repression of the Hes1 promoter. In addition, p65-NF{kappa}B is able to upregulate not only the Hes1 but also other promoters containing SRE and AP-1 sites, which are repressed by N-CoR. Thus, we conclude that p65-NF{kappa}B can regulate gene expression by a general mechanism that involves cytoplasmic translocation of the transcriptional corepressor protein N-CoR.

Key words: NF{kappa}B, N-CoR, Notch, Transcriptional regulation


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Notch/lin-12 family of transmembrane receptors plays important roles in regulating embryonic development and tissue homeostasis in the adult (reviewed in Artavanis-Tsakonas et al., 1999Go; Egan et al., 1998Go). Multiple observations suggest that Notch might be controlling different cellular events by inhibiting specific signals required for cell-type specification, cell-cycle progression and cell survival. When the Notch receptor binds to its ligand, which is located in neighboring cells, the intracellular domain of the protein (Notch-IC) is released from the cell membrane and translocated to the nucleus. There, active Notch (Notch-IC) interacts with the ubiquitous transcription factor RBPJ{kappa} to positively regulate gene transcription (reviewed in Egan et al., 1998Go). In fact, RBPJ{kappa} behaves as a bifunctional factor since, in the presence of Notch, it switches from being transcriptionally repressive to being transcriptionally active (Hsieh et al., 1996Go). Functional repression by RBPJ{kappa} has been described for different genes, including the adenovirus pIX polypeptide (Dou et al., 1994Go), Hes1 (Kao et al., 1998Go), IL-6 (Plaisance et al., 1997Go) or p52/NF{kappa}B2 (Oswald et al., 1998Go). Repression of the Hes1 promoter by RBPJ{kappa} has been associated with its interaction with the nuclear corepressors SMRT and N-CoR (Kao et al., 1998Go).

SMRT and N-CoR are highly homologous proteins, coded by two different genes with different splicing isoforms (Wong and Privalsky, 1998Go; Park et al., 1999Go). Although both proteins share many common structural and functional features, inactivation of the N-CoR gene by homologous recombination has shown that their functions are not physiologically redundant (Jepsen et al., 2000Go).

In recent years, coactivators and corepresors of nuclear receptors, and other transcription factors, have been identified as fundamental components in the regulation of eukaryotic gene expression (reviewed in Xu et al., 1999Go; Pazin and Kadonaga, 1997Go). Although transcriptional coactivator complexes possess intrinsic histone acetyl-transferase (HAT) activity, nuclear receptor corepressors achieve their function by recruiting histone deacetylases (HDACs). There is a positive correlation between core histone acetylation and gene transcriptional activity. It is generally accepted that acetylation causes local changes in chromatin structure, thus facilitating the assembly of the transcriptional machinery. Many transcription factors such as RBPJ{kappa} (Kao et al., 1998Go), NF{kappa}B, AP-1, SRF (Lee et al., 2000Go), MyoD (Bailey et al., 1999Go) and Pbx (Saleh et al., 2000Go) have been shown to regulate gene transcription by recruiting acetylase/deacetylase activities.

The NF{kappa}B/Rel family of transcription factors participates in the regulation of disparate cellular processes such as proliferation, differentiation, immune response and transcription of viral promoters (reviewed in Baldwin, 1996Go; Ghosh et al., 1998Go). The Rel family includes p65 (RelA), p105/p50, p100/p52, RelB, c-Rel and the viral oncoprotein v-Rel. These proteins associate as homo or heterodimers to form transcriptional regulatory complexes known as nuclear factor kappa B (NF{kappa}B). Transcriptional activity of NF{kappa}B is specified by the composition of the dimers and depends on their subcellular localization and their association with the inhibitory protein I{kappa}B (reviewed in Karin, 1999Go).

There is emerging evidence supporting a functional interplay between Notch and NF{kappa}B signaling pathways. For example, Notch can interact with p50-NF{kappa}B, thus modulating NF{kappa}B-dependent gene transcription (Guan et al., 1996Go). Conversely, NF{kappa}B induces the expression of the Notch ligand Jagged1, triggering Notch activation in adjacent cells (Bash et al., 1999Go). Interaction between both pathways is specially intriguing since NF{kappa}B can either promote or inhibit differentiation in various cell types (Kim et al., 2001Go; Feng and Porter, 1999Go; Kaliman et al., 1999Go; Kaisho et al., 2001Go) (L.E. and A.B, unpublished), whereas Notch activity is primarily involved in blocking this process (Fortini et al., 1993Go; Sternberg, 1988Go; Milner et al., 1996Go). We have now studied the putative effect of p65-NF{kappa}B on modulating the Notch/RBPJ{kappa} transcriptional activity. We describe here how the overexpression of p65-NF{kappa}B or a mutant that lacks the transcriptional activation (TA) domain facilitates Notch-IC-mediated transcription of the Hes1 promoter by inducing cytoplasmic retention of the nuclear corepressor N-CoR. This effect applies to other promoters repressed by N-CoR, such as those containing SRF or AP-1 sites. Further physiological studies are needed to understand the contribution of this new mechanism of gene regulation in the control of the different cellular events.


    Materials and Methods
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Plasmids
The Notch 1-IC (N1-IC) (AA 1807-2194) and Notch2-IC (N2-IC) (AA 1765-2154) cDNA expression vectors have been described previously (Bigas et al., 1998Go). Notch3-IC (N3-IC) (AA 1760-2070) was amplified by PCR, sequenced and cloned in the pCS2 vector in frame with the myc tag. Expression vectors for pCMV-flag-p65-NF{kappa}B, pCMV-I{kappa}B{alpha}32-36, pCDNA3-p50-NF{kappa}B, pCMV-flag-NcoR, pCMV-p65{Delta}TA, pCMV-p65{Delta}NES and reporter plasmids for Hes1-luc, 2x{kappa}B-luc, 2xSRE-luc 3xAP-1-luc have been previously described (DiDonato et al., 1996Go; Harhaj and Sun, 1999Go; Jarriault et al., 1995Go; Miralles et al., 1998Go).

Antibodies
Anti-flag (clone M2) was purchased from Sigma and used at a dilution of 1:1,000 for western blot and 1:750 for immunofluorescence analysis; anti-p65-NF{kappa}B (sc-109, Santa Cruz Biotechnology) was used at 1:400. 9E10, anti-myc-tag, was used at 1:1,000 for western blot and 1:200 for immunofluorescence. mN1A antibody (Huppert et al., 2000Go) was used 1:200 for western blot. Secondary antibodies conjugated with horseradish peroxidase (HRP) were purchased from DAKO and used 1:2,000 for western blot. Fluorescein-conjugated goat anti-mouse or Cy3-conjugated goat anti-rabbit (Amersham) secondary antibodies were diluted 1:200 and 1:1,000 respectively.

Cell culture and transfections
NIH-3T3 and 293T cells were cultured in Dulbecco's modified Eagle medium and 10% FBS (fetal bovine serum). Cells were plated at subconfluence and transfected by calcium phosphate. Medium was changed after 12 hours, and cells were processed 24 hours later for luciferase assays, immunofluorescence or western blots. Leptomycin B (LMB) was purchased from SIGMA and used at 10-40 ng/ml. Trichostatine A (TSA) was purchased from Calbiochem and used at 600 nM for 12 hours.

Western blot
293T cells were transfected with the different DNA plasmids by calcium phosphate treatment, and 48 hours later were lysed during 30 minutes at 4°C in a buffer containing 20% glycerol, 20 mM Hepes pH 7.6, 350 mM NaCl, 50 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.25% NP-40, 5 mM Na Fluoride, 1 mM EGTA, 0.25 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.4 mM Na-ortovanadate and 1 µg/ml Bestatin. Protein extracts were electrophoresed in 6% polyacrylamide gels and transferred to PVDF membranes overnight. Membranes were blocked with 5% non-fat dried milk in TBS and incubated with the appropriate antibody in TBS and 0.5% tween20 (TBS-T) with 5% non-fat dried milk for 90 minutes. Membranes were washed and incubated with a secondary HRP-conjugated antibody for 1 hour. After extensive washing, immunoreactive proteins were detected by using the Enhanced Chemiluminiscent Detection System (ECL, Amersham Pharmacia Biotech) as specified by the manufacturer.

Northern blot analysis
Total RNA was extracted from cells using the Chomczynski and Sacchi method (Chomczynski and Sacchi, 1987Go). RNAs were size-fractionated by electrophoresis, transferred onto Hybond-N+ nylon membranes (Amersham) and then hybridized with a radiolabeled Hes1 probe. Radioactivities of each signal were measured by a PhosphorImager using Quantity One software (Bio-rad). Densitometric analysis was performed by using Phoretics software.

Luciferase assays
NIH-3T3 were plated on 12-well plates and transfected with the indicated expression vectors or the empty vector as a control. In the different experiments we used 1 µg of Hes1-luc, 2x{kappa}B-luc, 3xAP-1-luc or 2xSRE-luc as reporter plasmids and 0.5 µg RSV-ß-gal as internal control. pCS2 vector was added when necessary to keep the amount of DNA constant. A luciferase assay (Luciferase Assay System, Promega) was performed 48 hours after transfection, following the manufacturer's instructions. Luciferase values were normalized for ß-galactosidase activity. At least three independent experiments were performed in duplicates.

Immunofluorescence
NIH-3T3 or 293T cells were seeded on slides at 20% confluence and transfected with 10 µg N-CoR, 4 µg p65-NF{kappa}B or 4 µg N1-IC. After 48 hours, cells were fixed in 3% paraformaldehyde in PBS for 25 minutes at 4°C, washed in PBS, permeabilized in 0.1% Triton X-100 in PBS, 5% non-fat dry milk for 25 minutes at 4°C. After washing, cells were incubated with the indicated primary antibody for 90 minutes at 4°C and extensively washed in PBS 1% non-fat dry milk. After 90 minutes of incubation with the appropriate secondary antibody, slides were extensively washed and mounted with Vectashield plus DAPI (Vector). Cells were visualized in an Olympus BX-60 microscope with the appropriate filters. Representative cells were photographed and slides were digitalized with Adobe Photoshop.


    Results
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
p65-NF{kappa}B synergizes with Notch to transactivate the Hes1 promoter in a p50-NF{kappa}B-independent manner
Notch-IC and NF{kappa}B are both involved in the control of cellular processes such as proliferation, differentiation and apoptosis. Previous observations showed that Notch-IC can modulate NF{kappa}B-regulated promoters both positively, by sequestering RBPJ{kappa} (Oswald et al., 1998Go), or negatively, by interacting with the p50-NF{kappa}B subunit (Guan et al., 1996Go). To investigate whether NF{kappa}B had a reciprocal effect on Notch/RBPJ{kappa}-regulated promoters, different Notch-IC and p65-NF{kappa}B expression vectors were cotransfected along with the Hes1-luc reporter plasmid into NIH-3T3 cells. Compared with Notch1-IC (N1-IC) alone, we observed that coexpression of full-length p65-NF{kappa}B (p65) increased the activity of the reporter gene in a dose-dependent manner (Fig. 1A) without affecting Notch-IC protein levels (Fig. 1A, lower panel). The maximum effect (two- to three-fold activation) was reached when equivalent amounts of Notch-IC and p65-NF{kappa}B (p65) were cotransfected. We found a similar increase when p65 was cotransfected with different activated Notch homologs: N1-IC (Fig. 1A), N2-IC (Fig. 1B) or N3-IC (Fig. 1C). In the absence of Notch-IC, no increase in the Hes1 transcriptional activity by p65-NF{kappa}B overexpression was observed, suggesting that there was no significant endogenous Notch activity in NIH-3T3 cells.



View larger version (16K):
[in this window]
[in a new window]
 
Fig. 1. Synergistic effect of p65-NF{kappa}B and Notch-IC in the transactivation of the Hes1 promoter. (A) N1-IC (0.5 µg) was cotransfected along with the Hes1-luc reporter (0.75 µg) in NIH-3T3 cells and increasing amounts of p65 wt. Lower panels show N1-IC and p65 protein levels. Protein loading for each lane was normalized according to ß-galactosidase expression. (B,C) p65-NF{kappa}B can synergize with different Notch-IC homologs. 0.5 µg of N2-IC (B) or N3-IC (C) were cotransfected with p65 wt (0.5 µg) along with the Hes1-luc reporter plasmid (1µg) in NIH-3T3 cells. (D) Synergistic effect of p65-NF{kappa}B and Notch-IC is not modified by ectopic expression of p50-NF{kappa}B. NIH-3T3 cells were cotransfected with different combinations of equivalent amounts (0.5 µg) of p50 subunit, N1-IC and p65 wt expression vectors. Luciferase activity is represented as the fold induction relative to the basal level measured in cells transfected with empty vector. The average and standard deviation of duplicates from a representative experiment are presented.

 

We next tested whether the previously reported interaction between Notch-IC and p50-NF{kappa}B (Guan et al., 1996Go) was responsible for the above-described effect. We hypothesized that addition of p65-NF{kappa}B would liberate Notch-IC from a putative p50 inhibition, resulting in an increase in Notch activity. Cotransfection of p50-NF{kappa}B did not affect the Notch activation of the Hes1 promoter nor the observed p65-mediated upregulation (Fig. 1D), although it increased NF{kappa}B-dependent transcription (data not shown). Altogether, these results demonstrate that p65-NF{kappa}B can synergize with Notch-IC to activate Hes1 transcription. Moreover, this effect is not due to a competition of both transcription factors for binding to the p50-NF{kappa}B subunit, indicating that other mechanisms may be involved.

Activation of NF{kappa}B-dependent transcription is not required for Notch and p65-NF{kappa}B synergism
Since NF{kappa}B participates in the transcriptional activation of several genes including the Notch ligand Jagged1 (Bash et al., 1999Go), we reasoned that the observed effect on the Hes1 promoter could be mediated by p65-NF{kappa}B transcriptional activation of any Notch pathway gene. To check this hypothesis, we repeated the p65/Notch-IC cotransfection experiments in the presence of the constitutive repressor of NF{kappa}B, I{kappa}B{alpha}32-36 (DiDonato et al., 1996Go). As expected, expression of I{kappa}B{alpha}32-36 inhibited p65-NF{kappa}B-mediated transactivation of the 2x{kappa}B-luc reporter in a dose-dependent manner (Fig. 2A) by sequestering NF{kappa}B in the cytoplasm even in the presence of Notch-IC (Fig. 2C). In these conditions, inhibition of NF{kappa}B transcriptional activity did not abrogate the p65-mediated upregulation of the Hes1 promoter (Fig. 2B). These results indicate that the synergistic effect of p65-NF{kappa}B and Notch on the Hes1 promoter requires neither colocalization of Notch and p65-NF{kappa}B in the nucleus nor NF{kappa}B transcriptional activity. To further demonstrate this observation, we coexpressed N1-IC with a deletion mutant of p65-NF{kappa}B (p65{Delta}TA), which lacks the transactivation domain (AA 451-551) (Harhaj and Sun, 1999Go). In the presence of N1-IC, expression of p65{Delta}TA resulted in the increased dose-dependent activation of the Hes1 promoter (Fig. 2D). Moreover, the effect of p65{Delta}TA was even stronger (up to four-fold) than the maximum effect observed with the p65 wild type (p65 wt).



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 2. Transcriptional activation of NF{kappa}B-dependent genes is not required for p65-mediated upregulation of the Hes1 promoter. NIH-3T3 cells were transfected with increasing amounts of I{kappa}B{alpha}32-36 in the presence of N1-IC (0.5 µg) and p65 wt (0.5 µg). The reporter constructs were (A) 2x{kappa}B-IL2-luc (1 µg) or (B) Hes 1-luc (1 µg). (C) Immunofluorescence assays to determine the subcellular localization of N1-IC and p65wt when coexpressed with I{kappa}B{alpha}32-36 in NIH-3T3 cells. Cells were cotransfected with N1-IC, p65 wt and I{kappa}B{alpha}32-36. N1-IC was detected with the 9E10 antibody and a FITC-labeled secondary antibody. p65 was detected with {alpha}-p65 and a Cy3-conjugated secondary antibody. (D) Luciferase fold activation obtained by cotransfecting the indicated amounts p65{Delta}TA with N1-IC (0.5 µg) and the Hes1-luc reporter (1 µg). Luciferase activity is presented as the fold induction relative to the basal level measured in cells transfected with empty vector. The average and standard deviation of duplicates from a representative experiment are presented. Lower panels show N1-IC and p65{Delta}TA protein levels. Protein loading for each lane was normalized according to ß-galactosidase expression.

 

Hes1 promoter activity is repressed by N-CoR, and this effect is reversed by ectopic expression of p65-NF{kappa}B
We have demonstrated that neither transcriptional activation by NF{kappa}B nor nuclear localization of p65 subunit are required for upregulating the Notch-dependent Hes1 promoter activation. Thus, we examined whether p65-NF{kappa}B was facilitating Notch activity by displacing a Hes1 repressor molecule.

As previously described for the SMRT corepressor (Kao et al., 1998Go), we found that N-CoR was able to repress Hes1 promoter activity in a dose-dependent manner (Fig. 3A). To investigate whether p65-NF{kappa}B was able to modulate N-CoR-mediated repression, we cotransfected N-CoR and increasing amounts of p65 wt or p65{Delta}TA along with the Hes1 promoter. Our results demonstrate that ectopic expression of p65wt (data not shown) or p65{Delta}TA were able to reverse the inhibitory effect of N-CoR in a dose-dependent manner (Fig. 3B). This suggests that the previously observed effect of p65-NF{kappa}B on the Hes1 promoter may be mediated through its interaction with N-CoR.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 3. The Hes1 promoter is repressed by N-CoR, and this effect is reversed by p65-NF{kappa}B overexpression. (A) N-CoR represses N1-IC activation of the Hes1 promoter. Indicated amounts of N-CoR were cotransfected with N1-IC expression vector (0.5 µg) into NIH-3T3 cells. (B) p65{Delta}TA can revert inhibition mediated by N-CoR. Increasing amounts of p65{Delta}TA were cotransfected with N1-IC (0.5 µg) and N-CoR (2 µg). Luciferase activity is presented as the fold induction relative to the basal level measured in cells transfected with empty vector. The average and standard deviation of duplicates from a representative experiment are presented. (C) Hes1 expression in 293T cells transfected with N2-IC and N-CoR expression vectors or coexpressed with p65wt is shown in the upper panel. Expression of N2-IC plasmid to compare transfection efficiency is shown in the lower panel.

 

We next asked whether overexpression of p65-NF{kappa}B was also modifying the expression of the endogenous Hes1 gene. We observed a moderate increase (1.5-fold by densitometric analysis) in the Hes1 mRNA from 293T cells transfected with Notch-IC and N-CoR in the presence of p65 wt (Fig. 3C) or p65{Delta}TA (data not shown).

p65{Delta}TA expression triggers cytoplasmic translocation of N-CoR
We then examined the subcellular localization and protein levels of N-CoR in the presence or absence of p65{Delta}TA. In the absence of p65{Delta}TA, N-CoR was localized in the nucleus, displaying a speckled distribution (upper panels), as previously reported (Horlein et al., 1995Go). However, in 293T cells cotransfected with N-CoR and p65{Delta}TA, we observed colocalization of both proteins in the cytoplasm, N-CoR being absent from the nucleus (Fig. 4A, lower panels). Identical results were obtained in NIH-3T3 cells (data not shown).



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 4. N-CoR is translocated to the cytoplasm in the presence of p65{Delta}TA. (A) Immunofluorescence staining of 293T cells transfected with N-CoR in the presence (lower panels) or absence (upper panels) of p65{Delta}TA. N-CoR-flag was detected by incubating with {alpha}-flag antibody and a FITC-conjugated secondary antibody (left panels), {alpha}-p65 staining is represented in the middle panels corresponding to Cy3 signal and nuclei were visualized by DAPI staining (right panels). Ectopic expression of p65{Delta}TA does not affect N-CoR protein levels. (B) Immunoblotting analysis of whole cell extracts isolated from 239T cells transfected with N1-IC and N-CoR-flag (lane 1) and N1-IC, N-CoR-flag and p65{Delta}TA (lane 2). Cell lysates were separated on a 6% acrylamide gel, and immunoblots were performed with the following antibodies: {alpha}-flag (N-CoR), {alpha}-N1(N1-IC) and {alpha}-p65. Protein loading for each lane was normalized according to ß-galactosidase expression and confirmed with {alpha}-N1 immunoblot. The western blot for p65 shows endogenous p65 (upper band) and transfected p65{Delta}TA (lower band).

 

We next quantified N-CoR protein levels in 293T cells cotransfected with N1-IC and N-CoR in the presence or absence of p65{Delta}TA (Fig. 4B). N-CoR was detected at comparable levels in both conditions, suggesting that protein stability was not affected.

p65-NF{kappa}B nuclear export is required for N-CoR cytoplasmic translocation and Hes1 upregulation
There is increasing evidence of continuous nuclear-cytoplasmic shuttling of p65-NF{kappa}B within the cell (Carlotti et al., 2000Go). p65-NF{kappa}B subcellular localization is regulated by its NES domain and by its interaction with the p50-NF{kappa}B subunit and I{kappa}B (Harhaj and Sun, 1999Go; Huang et al., 2000Go). It has previously been reported that incubation of cells with the CRM-1-mediated nuclear export inhibitor LMB (Nishi et al., 1994Go) results in nuclear accumulation of p65-NF{kappa}B (Huang et al., 2000Go). We next investigated whether inhibition of p65 nuclear export by LMB was blocking the cytoplasmic translocation of N-CoR. 293T cells were cotransfected with p65{Delta}TA and N-CoR and incubated in media alone or in the presence of 10 ng/ml of LMB. Fig. 5A shows that when CRM1-dependent nuclear export was inhibited, both p65{Delta}TA and N-CoR remained in the nucleus.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 5. p65-NF{kappa}B nuclear export is required for cytoplasmic retention of N-CoR. (A) Immunofluorescence staining of 293T cells transfected with N-CoR-flag and p65{Delta}TA in the absence (upper panel) or presence (lower panel) of LMB (10 ng/ml). (B) Immunofluorescence staining of 293T cells transfected with N-CoR-flag and p65{Delta}NES. N-CoR was detected with an {alpha}-flag antibody and a FITC-conjugated secondary antibody (left panels), {alpha}-p65 staining is represented in the middle panels corresponding to Cy3 signal, and nuclei were visualized by DAPI staining (right panels). (C) Hes1-luc transcriptional activity of NIH-3T3 cells cotransfected with N1-IC, N-CoR or/and p65{Delta}TA plasmids as indicated. Luciferase activity is represented as the fold induction relative to the basal level measured in cells transfected with empty vector. The average and standard deviation of duplicates from a representative experiment are presented. Lower panels show p65 protein levels. Protein loading for each lane was normalized according to ß-galactosidase expression.

 

To investigate whether p65 nuclear retention but not LMB incubation per se is responsible for inhibiting N-CoR cytoplasmic translocation, we used a p65 mutant (p65{Delta}NES) lacking the NES domain, which localizes exclusively in the nucleus (Harhaj and Sun, 1999Go). When coexpressed with p65{Delta}NES, N-CoR shows an exclusively nuclear localization (Fig. 5B). Consistent with this observation, increasing levels of p65{Delta}NES with N1-IC did not result in upregulation of Hes1 promoter (Fig. 5C). These results strongly suggest that p65 cytoplasmic translocation is required for N-CoR cytoplasmic retention and that this mechanism is responsible for p65-mediated Hes1 upregulation. Moreover, we have observed that incubation with the HDAC inhibitor trichostatine A (TSA) results in a more nuclear localization of p65-NF{kappa}B, as reported by Chen et al. (Chen et al., 2001Go). Nevertheless, the colocalization of N-CoR with p65-NF{kappa}B is not modified in these conditions, suggesting that deacetylase activity is not necessary for interactions between both proteins (data not shown).

p65-NF{kappa}B increases the activity of other promoters repressed by N-CoR
Repression of genes dependent on NF{kappa}B, Activator Protein-1 (AP-1) or Serum Response Factor (SRF) has been associated with the interaction of these transcription factors with SMRT (Lee et al., 2000Go). We next speculated that p65-induced N-CoR cytoplasmic translocation should also be affecting the expression of genes other than Hes1. We transfected N-CoR with or without p65{Delta}TA into NIH-3T3 cells and determined the transcriptional activity of SRF- and AP-1-dependent luciferase reporters. When incubated in 20% FBS, expression of p65{Delta}TA upregulates SRE reporter activity in a dose-dependent manner, up to a maximum of a three-fold increase (Fig. 6B). When the AP-1-dependent promoter was assayed in the presence of p65{Delta}TA, luciferase activity was five-fold higher compared with the maximum activity of this reporter in the presence of c-fos. (Fig. 6C). Moreover, and similar to its effect on the Hes1 reporter, we show that N-CoR was able to repress both promoters and that this repression was overridden by coexpression of p65{Delta}TA (Fig. 6A,B,C). These results clearly demonstrate that p65-NF{kappa}B can regulate gene expression not only through binding to promoters containing {kappa}B-sites but also by triggering cytoplasmic translocation of the nuclear corepressor N-CoR.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 6. p65{Delta}TA increases the activity of promoters repressed by N-CoR. NIH-3T3 cells were contransfected with the above indicated luciferase reporters (A). N1-IC (0.5 µg) and N-CoR (1 µg) were contransfected in the presence or absence of p65{Delta}TA (0.5 µg). Fold activation was calculated relative to cells transfected with N1-IC alone. (B) Cotransfection of p65{Delta}TA (0.5 µg) with or without N-CoR (1 µg) expression vector. Cells were incubated for 12 hours in the presence of 20% FBS, and fold activation was calculated relative to cells transfected with the reporter alone. (C) Cells were transfected with a c-fos expression vector (0.5 µg), p65{Delta}TA (0.5 µg), and/or N-CoR (1 µg). Fold activation was calculated relative to cells transfected with c-fos alone.

 


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Integration of different signal transduction pathways results in activation of specific subsets of genes and is responsible in the last term for specifying cell behavior. Notch and NF{kappa}B signaling pathways participate in the regulation of essential cellular events. In this study, we demonstrate that p65-NF{kappa}B enhances the transcriptional activity of Notch and that of other transcription factors by regulating the subcellular localization of the nuclear corepressor N-CoR.

First, we show that p65-NF{kappa}B cooperates with Notch in activating the Hes1 promoter. Second, we show that this effect is independent of transcriptional activity and colocalization of p65-NF{kappa}B and Notch-IC. Next, we demonstrate that ectopic expression of p65-NF{kappa}B can modify subcellular localization of the nuclear corepressor N-CoR. Finally, we demonstrate that this new mechanism affects not only Notch-dependent gene expression but also the expression of other genes (such as SRF- and AP-1-responsive genes) that are targets for N-CoR repression.

Notch and NF{kappa}B signaling pathways
Several interactions between Notch and NF{kappa}B pathways have already been reported; however, the physiological significance of their interplay is not well understood. For instance, a Notch-IC and p50-NF{kappa}B direct interaction leads to the inhibition of NF{kappa}B transcriptional activity, supporting the idea that Notch activation promotes downregulation of NF{kappa}B-dependent genes (Guan et al., 1996Go). Conversely, it has been shown that Notch3 homologue is able to upregulate the expression of NF{kappa}B-dependent genes, probably by promoting degradation of the NF{kappa}B inhibitor I{kappa}B{alpha} (Bellavia et al., 2000Go). On the other hand, NF{kappa}B participates in the transcriptional regulation of the Notch ligand Jagged1 gene (Bash et al., 1999Go), thus modifying the balance between receptors and ligands that is critical in Notch pathway regulation (Heitzler and Simpson, 1991Go).

Notch and NF{kappa}B can play different roles in regulating cell proliferation (Baonza and Garcia-Bellido, 2000Go; Carlesso et al., 1999Go; Kontgen et al., 1995Go), cell differentiation (Kaisho et al., 2001Go; Kaliman et al., 1999Go; Guttridge et al., 2000Go; Feng and Porter, 1999Go; Egan et al., 1998Go; Milner and Bigas, 1999Go) and apoptosis (reviewed in Barkett and Gilmore, 1999Go; Jehn et al., 1999Go; Shelly et al., 1999Go; Ohishi et al., 2000Go). However, it is tempting to speculate that they should exert antagonistic or synergistic effects depending on the cellular context. In myogenic systems, NF{kappa}B can both promote or inhibit differentiation depending on the external stimuli. For example, it mediates TNF-{alpha}-induced inhibition of differentiation in C2C12 cells (Guttridge et al., 1999Go), and it is required for the IGFII-induced differentiation of L6E9 myoblasts (Kaliman et al., 1999Go). In contrast, Notch activation is mainly associated with maintaining the undifferentiated phenotype (Kopan et al., 1994Go) by upregulating Hes1 (Kuroda et al., 1999Go), inhibiting MEF2C (Wilson-Rawls et al., 1999Go) or by combined mechanisms.

In T cell development, constitutive activation of Notch (Robey et al., 1996Go) and suppression of NF{kappa}B activity by expression of I{kappa}B{alpha}32-36 (Boothby et al., 1997Go) results in opposite phenotypes for the acquisition of the CD8+CD4- T cell fate. In fact, in a previous step, both Notch and NF{kappa}B activities are necessary for the survival of double-positive CD4+CD8+ cells when the appropriate TCR signal is received (Deftos et al., 2000Go; Hettmann and Leiden, 2000Go). Synergistic mechanisms of crosstalk between NF{kappa}B and Notch pathways (Oswald et al., 1998Go; Bash et al., 1999Go; Bellavia et al., 2000Go) (this paper) may be controlling thymocyte-positive selection by regulating the expression of anti-apoptotic genes. Consistent with this, both Notch and NF{kappa}B can activate bcl-2 expression in lymphoid cells (Zong et al., 1999Go; Deftos et al., 2000Go).

On the other hand, alterations in the Notch or NF{kappa}B pathways have been associated with oncogenic processes. Several cancer cells show constitutive nuclear NF{kappa}B activity, including those from lymphoid, breast, ovarian, lung, thyroid and melanoma origin (reviewed in Rayet and Gelinas, 1999Go). Moreover, there is emerging evidence that alteration of the Notch pathway affects many tumorigenic transformation and tumor progression processes (Capobianco et al., 1997Go; Leethanakul et al., 2000Go; Gallahan and Callahan, 1997Go). The NF{kappa}B constitutive activity found in Notch3-IC-transformed T cells is the first evidence that suggests a possible synergistic effect of both pathways in tumorigenesis (Bellavia et al., 2000Go). Taken together, these results indicate that coordination between Notch and NF{kappa}B pathways may be crucial in controlling cellular events.

Transcriptional activation mediated by p65-NF{kappa}B
Different combinations of Rel/NF{kappa}B subunits bind to specific target genes, directly regulating their transcriptional activity. Although NF{kappa}B-mediated transcriptional regulation has been extensively studied (reviewed in Siebenlist et al., 1994Go), NF{kappa}B-mediated effects on promoters lacking {kappa}b-sites or possible interactions of single NF{kappa}B subunits with other transcriptional complexes need further clarification.

For example, it has been reported that p65-NF{kappa}B can upregulate SRE-containing promoters independently of classical NF{kappa}B activity (Franzoso et al., 1996Go). In addition, the transforming ability of v-rel, a mutated version of the avian c-Rel that lacks the transactivation domain (reviewed in Luque and Gelinas, 1997Go), correlates with high levels of c-jun and c-fos proteins and increased AP-1 transcriptional activity (Kralova et al., 1998Go). Consistent with this, we have shown that overexpression of p65-NF{kappa}B regulates not only the transcriptional activity of Hes1 but also the activity of promoters containing SRE and AP-1 elements that lack NF{kappa}B sites. Thus, our results can provide an explanation of the previously published observations, reinforcing the idea that p65-NF{kappa}B exerts a positive effect over a broad spectrum of transcription factors, even in the absence of its transcriptional activation (TA) domain. In this sense, our results suggest that the p65{Delta}TA mutant may be more effective at overriding N-CoR transcriptional repression than the full-length protein, perhaps reflecting the presence of some regulatory elements in the C-terminal half of the protein.

Although more physiological studies should be done, these observations may reflect a new crosstalk between NF{kappa}B and other signaling pathways, which may be responsible for regulating transcription of specific sets of genes.

A role for p65-NF{kappa}B in regulating chromatin modifying activities
Assembly of multimeric protein complexes at their appropriate target DNA sequences results in transcriptional activation or repression, thus generating specific gene expression patterns (reviewed by Merika and Thanos, 2001Go). Recruitment of coactivators or corepresors by transcription factors and hormone receptors is crucial to determine the nature and activity of these complexes. Coactivators and corepressors exert their function by increasing or decreasing histone acetylation and thus modifying chromatin structure. N-CoR and SMRT nuclear corepressors are highly homologous, although N-CoR contains a unique N-terminal region that might be involved in unreported specific functions. Although preliminary studies associated both proteins with repression of hormone nuclear receptors (reviewed by Xu et al., 1999Go), SMRT can also repress c-fos-, NF{kappa}B-, SRF- (Lee et al., 2000Go) and Notch/RBPJ{kappa}-dependent genes (Kao et al., 1998Go). Here we show that most of these promoters are also repressed by N-CoR, and we demonstrate that this repression can be modulated by p65-NF{kappa}B.

Physical association of SMRT and HDACs with p65-NF{kappa}B has been reported to repress NF{kappa}B-regulated genes (Lee et al., 2000Go; Ashburner et al., 2001Go; Chen et al., 2001Go). Our results suggest that p65-NF{kappa}B may interact with N-CoR, inducing its translocation to the cytoplasm and acting as a rapid and potent tool for regulating gene expression. Although experiments with TSA suggest that this mechanism does not depend on deacetylase activity, the role of HDACs or other proteins known to interact with p65 or N-CoR is currently under investigation. It is also worth considering that phosphorylation or acetylation events affecting N-CoR or p65-NF{kappa}B may regulate their interaction. In this sense, it has been reported that SMRT can translocate to the cytoplasm in response to phosphorylation events (Hong and Privalsky, 2000Go; Jang et al., 2001Go).

Coactivators and corepressors bind to nuclear receptors competing for similar consensus regions (Nagy et al., 1999Go). Interestingly, p65-NF{kappa}B can synergize with IRF1 and ATF2/c-jun to activate transcription of the IFNß promoter by recruiting transcriptional coactivators through a leucine-rich domain (AA 430-458) similar to that present in nuclear receptors (Merika et al., 1998Go). In this system, the effect of p65-NF{kappa}B is dependent on the binding to DNA of all three transcription factors. By contrast, the mechanism that we describe here does not require the presence of a {kappa}B-binding site in the target promoter. Since the NES is included in the p65-NF{kappa}B leucinerich domain, it is tempting to speculate that there is a potential physical interaction between p65-NF{kappa}B and N-CoR involving this domain.

Taken together, our results indicate that p65-NF{kappa}B can exert an important role in coordinating gene transcription of specific sets of genes by a new mechanism that involves cytoplasmic translocation of N-CoR. We speculate that this new p65-NF{kappa}B function may be crucial in the integration of multiple signal transduction pathways.


    Acknowledgments
 
We would like to acknowledge M. Karin, J. Caamaño, R. Evans, S. Sun, A. Israel and H. Pahl for plasmids. We also thank L. A. Milner for the mN1A antibody. We are thankful to S. de la Luna and J. L. de la Pompa for critical reading and suggestions for the improvement of the manuscript, and H. Evans for helping in language correction, and FAPS for financial support for L.E. This work was supported by a grant (SAF98-052) from the Comisión Interministerial de Ciencia y Tecnología, Plan Nacional de Salud.


    References
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

Artavanis-Tsakonas, S., Rand, M. D. and Lake, R. J. (1999). Notch signaling: cell fate control and signal integration in development. Science 284,770 -776.[Abstract/Free Full Text]

Ashburner, B. P., Westerheide, S. D. and Baldwin, A. S., Jr. (2001). The p65 (RelA) subunit of NF-kappaB interacts with the histone deacetylase (HDAC) corepressors HDAC1 and HDAC2 to negatively regulate gene expression. Mol. Cell Biol. 21,7065 -7077.[Abstract/Free Full Text]

Bailey, P., Downes, M., Lau, P., Harris, J., Chen, S. L., Hamamori, Y., Sartorelli, V. and Muscat, G. E. (1999). The nuclear receptor corepressor N-CoR regulates differentiation: N-CoR directly interacts with MyoD. Mol. Endocrinol. 13,1155 -1168.[Abstract/Free Full Text]

Baldwin, A. S., Jr. (1996). The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu. Rev. Immunol. 14,649 -683.[Medline]

Baonza, A. and Garcia-Bellido, A. (2000). Notch signaling directly controls cell proliferation in the Drosophila wing disc. Proc. Natl. Acad. Sci. USA 97,2609 -2614.[Abstract/Free Full Text]

Barkett, M. and Gilmore, T. D. (1999). Control of apoptosis by Rel/NF-kappaB transcription factors. Oncogene 18,6910 -6924.[Medline]

Bash, J., Zong, W. X., Banga, S., Rivera, A., Ballard, D. W., Ron, Y. and Gelinas, C. (1999). Rel/NF-kappaB can trigger the Notch signaling pathway by inducing the expression of Jagged1, a ligand for Notch receptors. EMBO J. 18,2803 -2811.[Abstract/Free Full Text]

Bellavia, D., Campese, A. F., Alesse, E., Vacca, A., Felli, M. P., Balestri, A., Stoppacciaro, A., Tiveron, C., Tatangelo, L., Giovarelli, M. et al. (2000). Constitutive activation of NF-kappaB and T-cell leukemia/lymphoma in notch3 transgenic mice. EMBO J. 19,3337 -3348.[Abstract/Free Full Text]

Bigas, A., Martin, D. I. and Milner, L. A. (1998). Notch1 and Notch2 inhibit myeloid differentiation in response to different cytokines. Mol. Cell Biol. 18,2324 -2333.[Abstract/Free Full Text]

Boothby, M. R., Mora, A. L., Scherer, D. C., Brockman, J. A. and Ballard, D. W. (1997). Perturbation of the T lymphocyte lineage in transgenic mice expressing a constitutive repressor of nuclear factor (NF)-kappaB. J. Exp. Med. 185,1897 -1907.[Abstract/Free Full Text]

Capobianco, A. J., Zagouras, P., Blaumueller, C. M., Artavanis Tsakonas, S. and Bishop, J. M. (1997). Neoplastic transformation by truncated alleles of human NOTCH1/TAN1 and NOTCH2. Mol. Cell Biol. 17,6265 -6273.[Abstract]

Carlesso, N., Aster, J. C., Sklar, J. and Scadden, D. T. (1999). Notch1- induced delay of human hematopoietic progenitor cell differentiation is associated with altered cell cycle kinetics. Blood 93,838 -848.[Abstract/Free Full Text]

Carlotti, F., Dower, S. K. and Qwarnstrom, E. E. (2000). Dynamic shuttling of nuclear factor kappa B between the nucleus and cytoplasm as a consequence of inhibitor dissociation. J. Biol. Chem. 275,41028 -41034.[Abstract/Free Full Text]

Chen, L., Fischle, W., Verdin, E. and Greene, W. C. (2001). Duration of nuclear NF-kappaB action regulated by reversible acetylation. Science 293,1653 -1657.[Abstract/Free Full Text]

Chomczynski, P. and Sacchi, N. (1987). Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162,156 -159.[Medline]

Deftos, M. L., Huang, E., Ojala, E. W., Forbush, K. A. and Bevan, M. J. (2000). Notch1 signaling promotes the maturation of CD4 and CD8 SP thymocytes. Immunity 13, 73-84.[Medline]

DiDonato, J., Mercurio, F., Rosette, C., Wu-Li, J., Suyang, H., Ghosh, S. and Karin, M. (1996). Mapping of the inducible IkappaB phosphorylation sites that signal its ubiquitination and degradation. Mol. Cell Biol. 16,1295 -1304.[Abstract]

Dou, S., Zeng, X., Cortes, P., Erdjument-Bromage, H., Tempst, P., Honjo, T. and Vales, L. D. (1994). The recombination signal sequence-binding protein RBP-2N functions as a transcriptional repressor. Mol. Cell Biol. 14,3310 -3319.[Abstract]

Egan, S. E., St Pierre, B. and Leow, C. C. (1998). Notch receptors, partners and regulators: from conserved domains to powerful functions. Curr. Top. Microbiol. Immunol. 228,273 -324.[Medline]

Feng, Z. and Porter, A. G. (1999). NF-kappaB/Rel proteins are required for neuronal differentiation of SH-SY5Y neuroblastoma cells. J. Biol. Chem. 274,30341 -30344.[Abstract/Free Full Text]

Fortini, M. E., Rebay, I., Caron, L. A. and Artavanis-Tsakonas, S. (1993). An activated notch receptor blocks cell-fate commitment in the developing Drosophila eye. Nature 365,555 -557.[Medline]

Franzoso, G., Carlson, L., Brown, K., Daucher, M. B., Bressler, P. and Siebenlist, U. (1996). Activation of the serum response factor by p65/NF-kappaB. EMBO J. 15,3403 -3412.[Abstract]

Gallahan, D. and Callahan, R. (1997). The mouse mammary tumor associated gene INT3 is a unique member of the NOTCH gene family (NOTCH4). Oncogene 14,1883 -1890.[Medline]

Ghosh, S., May, M. J. and Kopp, E. B. (1998). NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu. Rev. Immunol. 16,225 -260.[Medline]

Guan, E., Wang, J., Laborda, J., Norcross, M., Baeuerle, P. A. and Hoffman, T. (1996). T cell leukemia-associated human Notch/translocation-associated Notch homologue has I kappa B-like activity and physically interacts with nuclear factor-kappa B proteins in T cells. J. Exp. Med. 183,2025 -2032.[Abstract]

Guttridge, D. C., Albanese, C., Reuther, J. Y., Pestell, R. G. and Baldwin, A. S. (1999). NF-kappaB controls cell growth and differentiation through transcriptional regulation of cyclin D1. Mol. Cell Biol. 19,5785 -5799.[Abstract/Free Full Text]

Guttridge, D. C., Mayo, M. W., Madrid, L. V., Wang, C. Y. and Baldwin, A. S. (2000). NF-kappaB-induced loss of MyoD messenger RNA: possible role in muscle decay and cachexia. Science 289,2363 -2366.[Abstract/Free Full Text]

Harhaj, E. W. and Sun, S. C. (1999). Regulation of RelA subcellular localization by a putative nuclear export signal and p50. Mol. Cell Biol. 19,7088 -7095.[Abstract/Free Full Text]

Heitzler, P. and Simpson, P. (1991). The choice of cell fate in the epidermis of drosophila. Cell 64,1083 -1092.[Medline]

Hettmann, T. and Leiden, J. M. (2000). NF-kappa B is required for the positive selection of CD8+ thymocytes. J. Immunol. 165,5004 -5010.[Abstract/Free Full Text]

Hong, S. H. and Privalsky, M. L. (2000). The SMRT corepressor is regulated by a MEK-1 kinase pathway: inhibition of corepressor function is associated with SMRT phosphorylation and nuclear export. Mol. Cell Biol. 20,6612 -6625.[Abstract/Free Full Text]

Horlein, A. J., Naar, A. M., Heinzel, T., Torchia, J., Gloss, B., Kurokawa, R., Ryan, A., Kamei, Y., Soderstrom, M., Glass, C. K. et al. (1995). Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor. Nature 377,397 -404.[Medline]

Hsieh, J. J., Henkel, T., Salmon, P., Robey, E., Peterson, M. G. and Hayward, S. D. (1996). Truncated mammalian Notch1 activates CBF1/RBPJk-repressed genes by a mechanism resembling that of Epstein-Barr virus EBNA2. Mol. Cell Biol. 16,952 -959.[Abstract]

Huang, T. T., Kudo, N., Yoshida, M. and Miyamoto, S. (2000). A nuclear export signal in the N-terminal regulatory domain of IkappaBalpha controls cytoplasmic localization of inactive NF-kappaB/IkappaBalpha complexes. Proc. Natl. Acad. Sci. USA 97,1014 -1019.[Abstract/Free Full Text]

Huppert, S. S., Le, A., Schroeter, E. H., Mumm, J. S., Saxena, M. T., Milner, L. A. and Kopan, R. (2000). Embryonic lethality in mice homozygous for a processing-deficient allele of Notch1. Nature 405,966 -970.[Medline]

Jang, M. K., Goo, Y. H., Sohn, Y. C., Kim, Y. S., Lee, S. K., Kang, H., Cheong, J. and Lee, J. W. (2001). Ca2+/calmodulin-dependent protein kinase IV stimulates nuclear factor-{kappa}B transactivation via phosphorylation of the p65 subunit. J. Biol. Chem. 276,20005 -20010.[Abstract/Free Full Text]

Jarriault, S., Brou, C., Logeat, F., Schroeter, E. H., Kopan, R. and Israel, A. (1995). Signalling downstream of activated mammalian Notch. Nature 377,355 -358.[Medline]

Jehn, B. M., Bielke, W., Pear, W. S. and Osborne, B. A. (1999). Protective effects of notch-1 on TCR-induced apoptosis. J. Immunol. 162,635 -638.[Abstract/Free Full Text]

Jepsen, K., Hermanson, O., Onami, T. M., Gleiberman, A. S., Lunyak, V., McEvilly, R. J., Kurokawa, R., Kumar, V., Liu, F., Seto, E. et al. (2000). Combinatorial roles of the nuclear receptor corepressor in transcription and development. Cell 102,753 -763.[Medline]

Kaisho, T., Takeda, K., Tsujimura, T., Kawai, T., Nomura, F., Terada, N. and Akira, S. (2001). IkappaB kinase alpha is essential for mature B cell development and function. J. Exp. Med. 193,417 -426.[Abstract/Free Full Text]

Kaliman, P., Canicio, J., Testar, X., Palacin, M. and Zorzano, A. (1999). Insulin-like growth factor-II, phosphatidylinositol 3-kinase, nuclear factor-kappaB and inducible nitric-oxide synthase define a common myogenic signaling pathway. J. Biol. Chem. 274,17437 -17444.[Abstract/Free Full Text]

Kao, H. Y., Ordentlich, P., Koyano-Nakagawa, N., Tang, Z., Downes, M., Kintner, C. R., Evans, R. M. and Kadesch, T. (1998). A histone deacetylase corepressor complex regulates the Notch signal transduction pathway. Genes Dev. 12,2269 -2277.[Abstract/Free Full Text]

Karin, M. (1999). How NF-kappaB is activated: the role of the IkappaB kinase (IKK) complex. Oncogene 18,6867 -6874.[Medline]

Kim, K. W., Kim, S. H., Lee, E. Y., Kim, N. D., Kang, H. S., Kim, H. D., Chung, B. S. and Kang, C. D. (2001). Extracellular signal-regulated kinase/90-KDA ribosomal S6 kinase/nuclear factor-kappa B pathway mediates phorbol 12-myristate 13-acetate-induced megakaryocytic differentiation of K562 cells. J. Biol. Chem. 276,13186 -13191.[Abstract/Free Full Text]

Kontgen, F., Grumont, R. J., Strasser, A., Metcalf, D., Li, R., Tarlinton, D. and Gerondakis, S. (1995). Mice lacking the c-rel proto-oncogene exhibit defects in lymphocyte proliferation, humoral immunity, and interleukin-2 expression. Genes Dev. 9,1965 -1977.[Abstract]

Kopan, R., Nye, J. S. and Weintraub, H. (1994). The intracellular domain of mouse Notch: a constitutively activated repressor of myogenesis directed at the basic helix- loop-helix region of MyoD. Development 120,2385 -2396.[Abstract/Free Full Text]

Kralova, J., Liss, A. S., Bargmann, W. and Bose, H. R. (1998). AP-1 factors play an important role in transformation induced by the v-rel oncogene. Mol. Cell Biol. 18,2997 -3009.[Abstract/Free Full Text]

Kuroda, K., Tani, S., Tamura, K., Minoguchi, S., Kurooka, H. and Honjo, T. (1999). Delta-induced Notch signaling mediated by RBP-J inhibits MyoD expression and myogenesis. J. Biol. Chem. 274,7238 -7244.[Abstract/Free Full Text]

Lee, S. K., Kim, J. H., Lee, Y. C., Cheong, J. and Lee, J. W. (2000). Silencing mediator of retinoic acid and thyroid hormone receptors, as a novel transcriptional corepressor molecule of activating protein-1, nuclear factor-kappaB, and serum response factor. J. Biol. Chem. 275,12470 -12474.[Abstract/Free Full Text]

Leethanakul, C., Patel, V., Gillespie, J., Pallente, M., Ensley, J. F., Koontongkaew, S., Liotta, L. A., Emmert-Buck, M. and Gutkind, J. S. (2000). Distinct pattern of expression of differentiation and growth-related genes in squamous cell carcinomas of the head and neck revealed by the use of laser capture microdissection and cDNA arrays. Oncogene 19,3220 -3224.[Medline]

Luque, I. and Gelinas, C. (1997). Rel/NF-kappa B and I kappa B factors in oncogenesis. Semin. Cancer Biol. 8,103 -111.[Medline]

Merika, M. and Thanos, D. (2001). Enhanceosomes. Curr. Opin. Genet. Dev. 11,205 -208.[Medline]

Merika, M., Williams, A. J., Chen, G., Collins, T. and Thanos, D. (1998). Recruitment of CBP/p300 by the IFN beta enhanceosome is required for synergistic activation of transcription. Mol. Cell 1,277 -287.[Medline]

Milner, L. A. and Bigas, A. (1999). Notch as a mediator of cell fate determination in hematopoiesis: evidence and speculation. Blood 93,2431 -2448.[Free Full Text]

Milner, L. A., Bigas, A., Kopan, R., Brashem-Stein, C., Bernstein, I. D. and Martin, D. I. (1996). Inhibition of granulocytic differentiation by mNotch1. Proc. Natl. Acad. Sci. USA 93,13014 -13019.[Abstract/Free Full Text]

Miralles, F., Parra, M., Caelles, C., Nagamine, Y., Felez, J. and Munoz-Canoves, P. (1998). UV irradiation induces the murine urokinase-type plasminogen activator gene via the c-Jun N-terminal kinase signaling pathway: requirement of an AP1 enhancer element. Mol. Cell. Biol. 18,4537 -4547.[Abstract/Free Full Text]

Nagy, L., Kao, H. Y., Love, J. D., Li, C., Banayo, E., Gooch, J. T., Krishna, V., Chatterjee, K., Evans, R. M. and Schwabe, J. W. (1999). Mechanism of corepressor binding and release from nuclear hormone receptors. Genes Dev. 13,3209 -3216.[Abstract/Free Full Text]

Nishi, K., Yoshida, M., Fujiwara, D., Nishikawa, M., Horinouchi, S. and Beppu, T. (1994). Leptomycin B targets a regulatory cascade of crm1, a fission yeast nuclear protein, involved in control of higher order chromosome structure and gene expression. J. Biol. Chem. 269,6320 -6324.[Abstract/Free Full Text]

Ohishi, K., Varnum-Finney, B., Flowers, D., Anasetti, C., Myerson, D. and Bernstein, I. D. (2000). Monocytes express high amounts of Notch and undergo cytokine specific apoptosis following interaction with the Notch ligand, Delta-1. Blood 95,2847 -2854.[Abstract/Free Full Text]

Oswald, F., Liptay, S., Adler, G. and Schmid, R. M. (1998). NF-kappaB2 is a putative target gene of activated Notch-1 via RBP-Jkappa. Mol. Cell. Biol. 18,2077 -2088.[Abstract/Free Full Text]

Park, E. J., Schroen, D. J., Yang, M., Li, H., Li, L. and Chen, J. D. (1999). SMRTe, a silencing mediator for retinoid and thyroid hormone receptors-extended isoform that is more related to the nuclear receptor corepressor. Proc. Natl. Acad. Sci. USA 96,3519 -3524.[Abstract/Free Full Text]

Pazin, M. J. and Kadonaga, J. T. (1997). What's up and down with histone deacetylation and transcription? Cell 89,325 -328.[Medline]

Plaisance, S., Vanden Berghe, W., Boone, E., Fiers, W. and Haegeman, G. (1997). Recombination signal sequence binding protein Jkappa is constitutively bound to the NF-kappaB site of the interleukin-6 promoter and acts as a negative regulatory factor. Mol. Cell. Biol. 17,3733 -3743.[Abstract]

Rayet, B. and Gelinas, C. (1999). Aberrant rel/nfkb genes and activity in human cancer. Oncogene 18,6938 -6947.[Medline]

Robey, E., Chang, D., Itano, A., Cado, D., Alexander, H., Lans, D., Weinmaster, G. and Salmon, P. (1996). An activated form of Notch influences the choice between CD4 and CD8 T cell lineages. Cell 87,483 -492.[Medline]

Saleh, M., Rambaldi, I., Yang, X. J. and Featherstone, M. S. (2000). Cell signaling switches HOX-PBX complexes from repressors to activators of transcription mediated by histone deacetylases and histone acetyltransferases. Mol. Cell. Biol. 20,8623 -8633.[Abstract/Free Full Text]

Shelly, L. L., Fuchs, C. and Miele, L. (1999). Notch-1 inhibits apoptosis in murine erythroleukemia cells and is necessary for differentiation induced by hybrid polar compounds. J. Cell. Biochem. 73,164 -175.[Medline]

Siebenlist, U., Franzoso, G. and Brown, K. (1994). Structure, regulation and function of NF-kappaB. Annu. Rev. Cell Biol. 10,405 -455.

Sternberg, P. W. (1988). Lateral inhibition during vulval induction in Caenorhabditis elegans.Nature 335,551 -554.[Medline]

Wilson-Rawls, J., Molkentin, J. D., Black, B. L. and Olson, E. N. (1999). Activated notch inhibits myogenic activity of the MADS-Box transcription factor myocyte enhancer factor 2C. Mol. Cell. Biol. 19,2853 -2862.[Abstract/Free Full Text]

Wong, C. W. and Privalsky, M. L. (1998). Transcriptional silencing is defined by isoform- and heterodimer-specific interactions between nuclear hormone receptors and corepressors. Mol. Cell. Biol. 18,5724 -5733.[Abstract/Free Full Text]

Xu, L., Glass, C. K. and Rosenfeld, M. G. (1999). Coactivator and corepressor complexes in nuclear receptor function. Curr. Opin. Genet. Dev. 9, 140-147.[Medline]

Zong, W. X., Edelstein, L. C., Chen, C., Bash, J. and Gelinas, C. (1999). The prosurvival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-kappaB that blocks TNFalpha-induced apoptosis. Genes Dev. 13,382 -387.[Abstract/Free Full Text]