NF-Y is essential for expression of the proapoptotic bim gene in sympathetic neurons

Hughes, R; Kristiansen, M; Lassot, I; Desagher, S; Mantovani, R; Ham, J

doi:10.1038/cdd.2010.166

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Original Paper
Published: 17 December 2010

NF-Y is essential for expression of the proapoptotic bim gene in sympathetic neurons

R Hughes¹,
M Kristiansen¹,
I Lassot^2,3,
S Desagher^2,3,
R Mantovani⁴ &
…
J Ham¹

Cell Death & Differentiation volume 18, pages 937–947 (2011)Cite this article

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Abstract

Neuronal apoptosis has a major role during development and aberrant apoptosis contributes to the pathology of certain neurological conditions. Studies with nerve growth factor (NGF)-dependent sympathetic neurons have provided important insights into the molecular mechanisms of neuronal apoptosis and the signalling pathways that regulate the cell death programme in neurons. The BH3-only protein Bim is a critical mediator of apoptosis in many cell types and in sympathetic neurons is required for NGF withdrawal-induced death. However, regulation of bim expression is complex and remains incompletely understood. We report that a conserved inverted CCAAT box (ICB) in the rat bim promoter is bound by the heterotrimeric transcription factor NF-Y. Interestingly, NF-Y is required for bim promoter activity and its induction following NGF withdrawal. We demonstrate that NF-Y activity is essential for endogenous Bim expression and contributes to NGF withdrawal-induced death. Furthermore, we find that the transcriptional coactivators CBP and p300 interact with NF-Y and FOXO3a and bind to this region of the bim promoter. The amount of CBP/p300 bound to bim increases after NGF deprivation and inhibition of CBP/p300 activity reduces bim induction. Our results indicate that NF-Y cooperates with FOXO3a to recruit CBP/p300 to the bim promoter to form a stable multi-protein/DNA complex that activates bim transcription after survival factor withdrawal.

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Main

The intrinsic (mitochondrial) pathway of apoptosis hinges on the balance of activity between proapoptotic and antiapoptotic members of the Bcl-2 superfamily. Bim is a BH3-only Bcl-2 family protein whose proapoptotic function is critical for apoptosis via the intrinsic pathway and Bim is one of just three BH3-only members that can bind to all five of their prosurvival relatives.¹ In addition, there is mounting evidence that Bim can induce mitochondrial outer membrane permeabilisation by binding to and directly activating the multidomain proapoptotic protein Bax.²

During development, sympathetic neurons depend on target-derived nerve growth factor (NGF) for their survival, and in its absence the cells die by apoptosis, usually within 24–48 h.³ Following NGF withdrawal, bim RNA and Bim protein levels increase rapidly and peak at around 16 h later.⁴ The overexpression of Bim_EL in sympathetic neurons is sufficient to induce the release of cytochrome c and apoptosis in the presence of NGF and sympathetic and sensory neurons isolated from bim^−/− knockout mice are significantly protected from trophic factor withdrawal-induced death.^{4, 5}

Because Bim is a potent inducer of apoptosis in many cell types, its expression is tightly regulated. In models of neuronal cell death, in which the cells depend on NGF for survival, three transcriptional pathways have been described. Activation of the MLK-JNK-c-Jun pathway is a key event for bim upregulation in sympathetic neurons: the overexpression of a dominant negative c-Jun protein reduces the increase in bim mRNA and protein level that occurs after NGF withdrawal⁴ and the jun^AA knock-in mutation, which eliminates the two major JNK phosphorylation sites in c-Jun, reduces the increase in Bim protein level after NGF withdrawal.⁶ Furthermore, bim transcription is negatively regulated by PI3-K-Akt signalling: active Akt phosphorylates FOXO3a, which is sequestered in the cytoplasm by 14-3-3 protein and following NGF withdrawal FOXO3a translocates into the nucleus of sympathetic neurons and activates bim expression via two conserved FOXO sites.⁷ Moreover, FOXO activity contributes to the NGF deprivation-induced death of sympathetic neurons.⁷ Additionally, bim is activated by a Cdk4-E2F-Myb pathway following NGF withdrawal in neuronally differentiated PC12 cells and this requires Myb-binding sites in the bim promoter.⁸ Previous studies have therefore identified important transcriptional mechanisms by which bim is regulated following growth factor withdrawal, however, nothing is currently known about the bim core promoter.

The DNA region that contains the core promoter is critical for the recruitment and assembly of the RNA polymerase II transcription initiation complex.⁹ It is important to fully understand the bim core promoter architecture, as the regulatory pathways that activate or repress bim transcription must ultimately act upon this region. Here, we use NGF-dependent sympathetic neurons to investigate how the DNA-binding transcription factor NF-Y and the coactivators CBP and p300 contribute to bim promoter function and activation following NGF withdrawal. Our results suggest a model in which NF-Y and CBP/p300, together with FOXO3a, are part of a multi-protein complex that is regulated by NGF.

Results

NF-Y binds to an inverted CCAAT box in the bim proximal promoter

Since bim has a TATA-less promoter we were interested to establish what factor(s) control its basal promoter function and how these regulate bim expression following NGF deprivation in sympathetic neurons. Using ConSite DNA analysis software¹⁰ (http://www.phylofoot.org/) we identified a conserved inverted CCAAT box (ICB)¹¹ that is located 29 or 54 bp upstream of the major transcriptional start site in rat and human bim, respectively (Figure 1). The major CCAAT box-binding protein is NF-Y, a heterotrimer formed by the interaction of three subunits, NF-YA, B and C.¹¹

To investigate whether NF-Y can bind to the ICB in the bim promoter, we performed some electrophoretic mobility shift assays (EMSAs). An oligo containing a previously identified CCAAT box in the rat GTP cyclohydrolase I (GTP C1) gene¹² was used as a positive control (Figure 2a, lanes 1–4). Initially EMSA experiments were performed with nuclear extracts from neuronally differentiated PC6-3 cells, a PC12 subline (Figure 2a). The nuclear extracts contained proteins that bound the GTP C1 CCAAT box (lanes 1–4) and this binding (marked NF-Y complex) was abolished by point mutations in the CCAAT box consensus (lanes 5 and 6). NF-YA is the regulatory subunit and is responsible for sequence specific DNA binding.¹³ Therefore, antibodies specific to the NF-YA subunit or to c-Jun (as a negative control) were used for supershift assays. The c-Jun antibody did not alter the binding pattern (lane 4), whereas the NF-Y antibody supershifted the entire protein complex (marked ss) (lane 3). The binding of NF-Y to the bim ICB (lane 8) was similar to the binding of NF-Y to the GTP C1 probe (lane 2), and the NF-Y complex behaved in an identical fashion when antibodies against NF-Y were added (lanes 3 and 9). We also performed EMSA experiments with whole cell extracts prepared from sympathetic neurons (Figure 2b). Extracts from sympathetic neurons maintained in the presence of NGF (lane 2) reproduced the binding pattern that was observed with extracts from differentiated PC6-3 cells in Figure 2a, and binding did not change following NGF withdrawal (lane 3). The NF-Y complex was supershifted with the NF-YA antibody (lanes 4 and 5) but not a negative control antibody (Fra-2; lanes 6,7) and binding was abolished by mutations in the ICB (lanes 9 and 10).

To confirm that NF-Y can bind to the endogenous bim promoter, we carried out chromatin immunoprecipitation (ChIP) assays using the NF-YA antibody or with an antibody against MyoD as a negative control (Figures 2c and d). Neuronally differentiated PC6-3 cells were maintained in the presence or absence of NGF for 16 h. The binding of NF-Y to the ICB in the bim promoter was detected by PCR, using primers that amplify the region that contains the ICB. The negative control antibody to MyoD immunoprecipitated background levels of bim promoter chromatin, however this was significantly enriched by the antibody against NF-YA in cells maintained in the presence of NGF and following NGF withdrawal (Figures 2c and d). Therefore, NF-Y is bound to the ICB in the rat bim promoter in the presence and absence of NGF, with no significant change in the amount of NF-Y bound to the promoter following withdrawal of NGF.

The ICB is critical for bim promoter function and contributes to the activation of bim transcription following NGF withdrawal

A bim-LUC reporter construct is significantly activated after NGF withdrawal in microinjected sympathetic neurons.^{7, 14} To determine whether the ICB is important for bim promoter activity, the point mutations that abolished NF-Y binding (Figures 2a and b) were introduced into the ATTGG pentamer within the bim-LUC construct (Figure 3a). The bim-LUC or bim-LUC CCAAT (−) constructs were then microinjected into sympathetic neurons and luciferase activity was determined following 16 h in the presence or absence of NGF. Mutation of the ICB resulted in an 80% decrease in basal promoter activity (Figure 3b). Following NGF withdrawal the bim-LUC reporter was activated significantly as previously reported.^{7, 14} The basal levels of the two constructs, in the presence of NGF, were then set to one and the induction factors of the constructs compared (Figure 3c). Mutation of the ICB significantly decreased the fold induction of the bim promoter following NGF withdrawal, from 2.37±0.19 to 1.50±0.13.

NF-Y activity is required for Bim expression and contributes to NGF withdrawal-induced death

To establish whether NF-Y activity is required for bim promoter function, sympathetic neurons were microinjected with the YA13 m29 expression vector along with guinea pig IgG (gp IgG) as a marker (Figure 4). YA13 m29 is a dominant negative mutant of the NF-YA subunit.¹⁵ It can bind to the other NF-Y subunits but not to DNA and thus acts by forming inactive NF-Y trimers. The YA13 m29 construct was microinjected into sympathetic neurons and after 24 h the cells were stained with an anti-NF-YA antibody. NF-YA immunostaining was considerably brighter in the nuclei of injected cells (Figure 4a) and over 90% of the injected neurons expressed high levels of NF-YA. Sympathetic neurons were then co-microinjected with YA13 m29, or the empty vector pSG5, along with the bim-LUC construct (Figure 4b). Expression of YA13 m29 completely inhibited the induction of bim-LUC following NGF withdrawal and also caused a significant decrease in the basal level of bim-LUC.

We then studied the effect of YA13 m29 on endogenous Bim protein levels (Figures 4c and d). Sympathetic neurons were microinjected with the YA13 m29 construct, or pSG5, and then cultured ±NGF for 16 h, after which time the cells were fixed and stained with an anti-Bim antibody. Bim is present in the cytoplasm at low levels in the presence of NGF, but strongly increases in level following NGF withdrawal (Figure 4c). Following NGF deprivation, almost 60% of the cells injected with pSG5 expressed high Bim levels (Figure 4d). However, when the cells were injected with the YA13 m29 construct the percentage of cells expressing high levels of Bim was significantly reduced, to 10% (Figure 4d).

To investigate the role of NF-Y in NGF withdrawal-induced death, sympathetic neurons were microinjected with the YA13 m29 construct, or pSG5, together with Texas red dextran (TRD) as a marker. The neurons were then maintained in the presence or absence of NGF and the number of viable injected cells was counted at 0, 24, 48 and 72 h. Viable neurons were morphologically normal and retained TRD in the nucleus (Figure 5a). At 72 h following NGF withdrawal, the number of viable cells injected with the pSG5 control was below 20% (Figure 5b), whereas cells injected with YA13 m29 were partially protected from cell death (39% were viable) (Figure 5b). The injected neurons not protected by YA13 m29 died by apoptosis following NGF withdrawal (Supplementary Figure S1). These results indicate that NF-Y activity is necessary for cell death in NGF-deprived sympathetic neurons.

CBP and p300 are important for activation of the bim promoter following NGF withdrawal

The ICB and NF-Y activity are necessary for bim promoter activation following NGF withdrawal, but the mechanism by which NF-Y functions has yet to be elucidated, since NF-Y is bound to the ICB in both the presence and absence of NGF. CBP and p300 are two distinct but highly homologous transcriptional coactivators. They can activate the transcription of a multitude of target genes and can interact with over 350 viral and mammalian proteins¹⁶ including c-Jun, FOXO3a, Myb and NF-Y, which activate bim transcription in sympathetic neurons.^{4, 7, 8} We therefore investigated whether CBP/p300 are important for bim promoter activation following NGF withdrawal. In immunocytochemistry experiments, we confirmed that CBP and p300 localise to the nucleus of sympathetic neurons, with ∼95% of sympathetic neurons expressing high levels of CBP and p300 in the presence and absence of NGF (Supplementary Figure S2). To study whether CBP and p300 are required for bim promoter activation, antibodies specific for CBP or p300 were co-injected into sympathetic neurons together with the bim-LUC construct (Figure 6a). The microinjection of specific antibodies can be used as a suitable alternative to conventional protein knockdown techniques in these cells.^{17, 18, 19} The bim-LUC construct was not significantly activated following NGF withdrawal when antibodies against CBP or p300 were co-injected, compared with the control rabbit IgG (Figure 6a). We also performed a positive control antibody co-injection experiment using a CREB reporter construct pCRE-LUC (Figure 6b), which requires CBP and p300 for its activation by protein kinase A and CREB. Sympathetic neurons were co-injected with pCRE-LUC together with the CBP or p300 antibodies or rabbit IgG. Following injection the cells were treated with CPTcAMP, a membrane permeable cAMP analogue. The pCRE-LUC construct was significantly activated by CPTcAMP treatment when co-injected with IgG compared with the control construct without CRE sites, p-LUC. Importantly, injection of the CBP or p300 antibodies significantly decreased this induction.

Studies with p300 and CBP knockout mice indicate that CBP and p300 are present in cells at limiting concentrations.^{20, 21} Therefore, overexpression of p300 or CBP should activate the bim promoter in the presence of NGF. Sympathetic neurons were co-microinjected with pRc/RSV-mCBP or the control empty vector pBJ9Ω, or CMVp300 or the control empty vector pcDNA3.1, together with the bim-LUC reporter construct (Figure 6c). Bim-LUC was significantly activated by overexpression of either CBP or p300. Interestingly, overexpression of p300 did not activate bim-LUC CCAAT (−) (Figure 6d) suggesting that the ICB must be intact for activation of the bim promoter by p300.

The amount of CBP/p300 bound to the bim promoter is regulated by NGF withdrawal

To confirm that p300 and CBP can bind to the endogenous bim promoter, we performed ChIP assays using differentiated PC6-3 cells and the p300 and CBP antibodies, along with antibodies against MyoD and Bim as negative controls (Figures 7a and b). The binding of p300 or CBP to the bim core promoter was studied by PCR using the primers that were previously used to study the binding of NF-Y to the ICB. In the presence of NGF, there was some binding of p300 and CBP to the bim promoter but this was significantly enriched following NGF withdrawal (Figures 7a and b). Therefore, p300 and CBP are bound to the same region of the bim promoter as NF-Y and the amount of p300 and CBP bound to the promoter increases ∼2.1-fold following NGF withdrawal.

CBP/p300 interact with NF-Y in the presence of NGF and following NGF withdrawal

To investigate whether NF-Y and CBP/p300 interact in extracts from NGF-dependent cells, co-immunoprecipitation (co-IP) experiments were carried out (Figure 7c and Supplementary Figure S3a). Nuclear extracts were prepared from differentiated PC6-3 cells cultured ±NGF for 16 h. The nuclear extracts were immunoprecipitated with antibodies against p300, CBP, NF-YA or rabbit IgG. Immunoblotting was then performed using an NF-YA antibody (Figure 7c). Importantly, NF-YA was precipitated with antibodies against CBP and p300 but not the control rabbit IgG. In addition, the NF-Y antibody immunoprecipitated p300 and CBP (Supplementary Figure S3a). These results demonstrate that NF-Y interacts with CBP and p300 in the nucleus of PC6-3 cells cultured in the presence or absence of NGF.

Following NGF withdrawal FOXO3a binds to the bim promoter and interacts with CBP/p300

Since FOXO3a contributes to the induction of bim transcription after NGF withdrawal⁷ we investigated whether, like NF-Y, FOXO3a bound to CBP and p300, as reported in other systems.²² First, we performed ChIP assays on differentiated PC6-3 cells using a FOXO3a antibody and MyoD and Bim antibodies as negative controls (Figures 7e and f and Supplementary Figures S3c and d). The binding of FOXO3a to FOXO site 1 (located upstream of the ICB) and FOXO site 2 (located at the exon 1/intron junction) was studied by PCR. Following NGF withdrawal there was a large increase (∼4.5 fold) in the amount of FOXO3a bound to each site (Figures 7e and f and Supplementary Figures S3c and d).

We then investigated in co-IP experiments whether FOXO3a can interact with CBP/p300 in nuclear extracts prepared from differentiated PC6-3 cells cultured ±NGF (Figure 7d and Supplementary Figure S3b). The lysates were immunoprecipitated with antibodies against p300, CBP, FOXO3a or rabbit IgG. Immunoblotting was then performed using a FOXO3a antibody (Figure 7d). In −NGF extracts, FOXO3a was precipitated with antibodies against CBP and p300, but this was not the case in the presence of NGF, or in either condition for the control rabbit IgG. In the reverse experiment, CBP and p300 were precipitated with the FOXO3a antibody, but again only after NGF withdrawal (Supplementary Figure S3b).

The bim promoter is not activated in response to NGF withdrawal following the combined loss of NF-Y and FOXO binding sites

To assess the relative contribution of the FOXO and NF-Y binding sites in the bim promoter, we mutated both FOXO sites and the ICB within bim-LUC (Figure 8a). We compared bim-LUC CCAAT(−) FOXO(dm) with the wild type bim-LUC in microinjection experiments with sympathetic neurons (Figure 8b). Loss of both FOXO sites, together with the ICB, significantly reduced the basal promoter level, as shown previously for bim-LUC CCAAT (−). Interestingly, loss of all three sites completely abolished the induction of bim-LUC following NGF withdrawal. This indicates that the two FOXO sites are required for the residual induction that is seen with bim-LUC CCAAT (−) after NGF deprivation.

We also investigated the effect on cell survival after NGF withdrawal of inhibiting both NF-Y and FOXO activity. YA13 m29 was co-expressed with the FKH (DBD), and TRD was used as a marker. The FKH (DBD) construct expresses the DNA-binding domain (DBD) of FOXO3a, which is a specific inhibitor of FOXO transcriptional activity.⁷ Following injection, the neurons were withdrawn from NGF for 72 h and the number of viable injected cells was determined (Figure 8c). In accordance with Figure 5b, at 72 h following NGF withdrawal, the number of viable cells injected with the control (pSG5+pcDNA.1) was ∼15%, whereas cells that were injected with YA13 m29 were partially protected from cell death (∼40% of cells were viable at 72 h −NGF). As demonstrated previously by Gilley et al.,⁷ neurons injected with the FKH (DBD) construct were better protected (58% of cells were viable at 72 h −NGF). Co-expression of both YA13 m29 and FKH (DBD) did not significantly increase cell survival compared with injection of the FKH (DBD) construct alone (Figure 8c). This is probably because the FKH (DBD) construct alone already gives the maximum protection observed in this microinjection assay, similar to that conferred by Bcl-2, dominant negative c-Jun or the JNK binding domain of JIP-1.^{23, 24}

Discussion

We have studied the role of a conserved inverted CCAAT box in the bim core promoter that is bound by NF-Y. This is the first bim promoter element that has been shown to be important for basal promoter function and as the bim promoter does not contain a TATA box, it is likely that the NF-Y-binding-ICB has an important role in pre-initiation complex assembly as reported for other TATA-less promoters.²⁵ In addition, the ICB is critical for bim promoter activation following NGF withdrawal.

We used the YA13 m29 dominant negative mutant^{15, 26, 27} to establish that NF-Y activity is required for bim promoter function and expression of the endogenous Bim protein. Importantly, we also demonstrated that expression of YA13 m29 partially protects sympathetic neurons from NGF withdrawal-induced death (∼40% of the cells are viable at 72 h −NGF). This level of protection is similar to the effect of a complete bim^−/− knockout on neuronal survival.^{4, 5} This partial protection is likely to be because the other BH3-only proteins that increase in level after NGF withdrawal, Puma and Dp5, can partially compensate for the loss of Bim.^{28, 29, 30}

NF-Y can recruit the transcriptional cofactors CBP and p300 to DNA to potentiate transcription.³¹ As the amount of NF-Y bound to the bim promoter does not increase after NGF withdrawal, we hypothesised that the recruitment of transcriptional cofactors to the bim promoter may be regulated by NGF withdrawal. We found that co-injection of antibodies against CBP or p300 inhibits the induction of bim-LUC after NGF withdrawal and that CBP and p300 bind to the region of the bim promoter that is bound by NF-Y, and that the amount of CBP/p300 associated with bim significantly increases after NGF withdrawal.

Since CBP and p300 do not bind DNA directly,³² they must interact with proteins that are already bound to the bim promoter. We have shown that mutation of the ICB abolishes the activation of the bim-LUC reporter by p300 and that NF-Y interacts with CBP and p300 in extracts from neuronal cells. This suggests that CBP/p300 may activate the bim promoter through protein–protein interactions with NF-Y bound to the ICB. Furthermore, studies in other systems have demonstrated that CBP/p300 can interact with the other regulatory factors, c-Jun, FOXO and Myb,^{22, 33, 34} which activate bim transcription in neurons. We have shown that FOXO3a binds to two conserved FOXO sites in the bim promoter following NGF withdrawal. In addition, we have demonstrated that FOXO3a interacts with CBP and p300 in extracts from neuronal cells, in the absence of NGF.

CBP and p300 can act as a ‘bridge’ to connect sequence-specific transcription factors to the basal transcription machinery³² and their large size allows multiple proteins to bind to them simultaneously. It is possible that a multi-protein enhanceosome-like structure³⁵ may form on the bim promoter following NGF withdrawal, since we have shown that CBP/p300 can bind to NF-Y and FOXO3a, and NF-Y is known to recruit the basal transcription machinery and RNA polymerase II to the promoters of CCAAT box-containing genes.²⁵ It will be interesting to establish whether this mechanism is also critical in other cell types as the ICB is common to the rat, mouse and human bim genes and NF-Y, FOXO3a and CBP/p300 are widely expressed. Preliminary experiments using mouse cerebellar granule neurons (CGNs) suggest that the ICB may be important for bim transcription in these cells (Supplementary Figure S4).

In summary, our results support a model (Figure 9) in which NF-Y is bound to the ICB in the presence of NGF and recruits some CBP/p300 to the bim promoter. In sympathetic neurons deprived of NGF, FOXO3a translocates from the cytoplasm into the nucleus and binds to the two conserved FOXO sites that flank the ICB.⁷ CBP/p300 binding to the bim promoter increases significantly after NGF withdrawal because CBP/p300 can be recruited by FOXO3a as well as NF-Y. Therefore, following NGF withdrawal, NF-Y and FOXO3a (and potentially other factors such as c-Jun and Myb) recruit CBP/p300 to form a stable multi-protein complex that increases the rate of bim transcription (Figure 9). Such protein–protein interactions would therefore integrate the different signalling pathways that cooperatively activate the bim promoter following survival factor withdrawal.

Materials and Methods

Plasmid constructs

The bim-LUC reporter consists of a 5.2 kb fragment, containing the region immediately 5′ to the rat bim initiator codon, sub-cloned into pGL3-basic.⁷ The bim-LUC CCAAT(−) construct was generated using the oligonucleotides 5′-GAGCCCGCGGTGAGCTCGCGCCGGAGCGGG-3′ and 5′-CCCGCTCCGGCGCGAGCTCACCGCGGGCTC-3′. The altered bases (underlined) create four point mutations in the ICB. The bim-LUC CCAAT (−) FOXO(dm) construct was generated by incorporating point mutations into FOXO site 1 and FOXO site 2 within bim-LUC CCAAT (−) using oligonucleotides described previously.⁷ Mutagenesis was carried out using the QuikChange II XL site-directed mutagenesis kit (Stratagene, Agilent, Santa Clara, CA, USA). YA13 m29 is a dominant negative mutant of the NF-YA subunit¹⁵ cloned into the expression vector pSG5 (Stratagene). The expression vector for p300 was supplied by Upstate (Millipore, Billerica, MA, USA). It consists of the human p300 cDNA cloned into pCMVβ (CMVp300). The expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA, USA), which contains the CMV promoter, was used as an empty vector control. The expression vector for CBP was kindly provided by Professor Tony Kouzarides (The Gurdon Institute, Cambridge, UK). It contains the mouse CBP cDNA cloned into pRc/RSV (pRc/RSV-mCBP). The expression vector pBJ9Ω, which is also under the control of the RSV promoter, was used as an empty vector control (also supplied by T Kouzarides). The CREB reporter construct pCRE-LUC and the control without CRE sites, pLUC, were supplied by Stratagene. FKH (DBD)⁷ is the DNA-binding domain of FOXO3a cloned into the expression vector pcDNA1 (Invitrogen).

Cell culture

Sympathetic neurons were isolated from the superior cervical ganglia (SCG) of 1-day-old Sprague Dawley rats and cultured as described previously.³⁶ Cells were maintained in SCG medium supplemented with 2.5S NGF (Cedarlane, Burlington, Canada) at 50 ng/ml, and fluorodeoxyuridine and uridine (both from Sigma-Aldrich, St Louis, MO, USA), each at 20 μ M. PC6-3 cells, a subline of the PC12 cell line,³⁷ were cultured as described previously.¹⁹ For differentiation, PC6-3 cells were maintained in medium containing 2% horse serum, 1% FCS and NGF (Promega, Fitchburg, WI, USA) at 100 ng/ml, for 5–7 days. In NGF withdrawal experiments, cells were rinsed twice with medium (without NGF) and then refed with medium containing an anti-NGF antibody at 100 ng/ml (Chemicon, Millipore). For experiments with pCRE-LUC and pLUC, sympathetic neurons were refed with +NGF medium containing 500 μ M CPTcAMP (Sigma-Aldrich).

Cerebellar granule neurons were prepared from 7-day-old mice and maintained in medium containing 25 mM KCl, as described previously.^{38, 39} In KCl deprivation experiments, neurons were washed once and then incubated in serum-free BME supplemented with L-Gln, HEPES, antibiotics and 1 μ M of the NMDA antagonist (+) MK-801, and 5 mM KCl (K5 medium) to induce apoptosis.

Microinjection

Sympathetic neurons were cultured in vitro for 5–7 days and then microinjected as described previously.⁴ Approximately 150 cells were injected per coverslip and each injection experiment was repeated at least three times. Plasmids were injected into the nucleus of each cell and over 90% of cells survived injection.

Dual luciferase assays were carried out on injected cells using the Dual luciferase reporter assay system (Promega). Cells were harvested for luciferase assays at 16 h following NGF withdrawal.

For antibody coinjection experiments, the Santa Cruz (Santa Cruz, CA, USA) p300 (C-20:Sc-585 X) and CBP (C-20:Sc-583 X) antibodies were diluted in PBS (−) and centrifuged in Microcon YM-3 centrifugal filters (Millipore) to remove sodium azide. Antibodies were injected at a final concentration of 1 μg/μl and purified rabbit immunoglobulin in PBS (Jackson ImmunoResearch, West Grove, PA, USA) was used as a control.

In cell survival assays, neurons were injected with the YA13 m29 expression vector or the control empty vector (pSG5) at 200 ng/μl, along with 5 μg/μl of TRD (70 000 MW; Molecular Probes, Invitrogen, Carlsbad, CA, USA).

Transfection of CGNs

CGNs cultured in 24-well plates were transfected at 5 days in vitro with 0.5 μg of pRL-TK together with 1.5 μg of either the bim-LUC or bim-LUC CCAAT(−) constructs, using calcium phosphate precipitation as described previously.³⁹ After 24 h the cells were either left untreated (K25) or deprived of KCl (K5). Four hours after deprivation, transfected neurons were harvested for luciferase assays using the Dual luciferase reporter assay system.

Immunocytochemistry

Neurons were injected with the YA13 m29 expression vector or the control empty vector (pSG5) at 20 ng/μl along with 2.5 μg/μl of purified gp IgG (Jackson ImmunoResearch) and fixed and stained as described before.⁷ In brief, cells were fixed in 4% paraformaldehyde, permeabilised in 0.5% Triton-X-100 and blocked in 50% goat serum (Sigma-Aldrich). After washing, cells were incubated in a primary antibody to detect NF-YA (Santa Cruz Sc-10779:H-209) or Bim (# 2819; Cell Signalling, Danvers, MA, USA). Secondary antibodies were a FITC-conjugated anti-rabbit IgG, and a rhodamine anti-gp IgG if neurons were microinjected (both from Jackson ImmunoResearch). Secondary-only controls were carried out to confirm the specificity of primary antibodies and cells were scored in a blinded manner whenever possible. TUNEL analysis was carried out using an in situ cell death detection kit (Roche, Basel, Switzerland). All slides were viewed using a Zeiss Axioplan 2 fluorescence microscope.

Electrophoretic mobility shift assay

EMSA experiments were performed on nuclear lysates from differentiated PC6-3 cells maintained in medium containing NGF or withdrawn from NGF for 16 h, and whole cell lysates from sympathetic neurons maintained in medium containing NGF or withdrawn from NGF for 16 h. Nuclear lysates were prepared from PC6-3 cells and whole cell extracts were prepared from sympathetic neurons as described previously.^{19, 40} Double-stranded oligonucleotides with 5′ overhangs were labelled by filling in with Klenow polymerase (Roche) and (α-³²P) dCTP (3000 Ci/mmol; PerkinElmer, Wellesley, MA, USA). Oligonucleotide pairs used for EMSA experiments were as follows: bim CCAAT wt 5′-CTAGCCCGCGGTGATTGGGCGCCGGAG-3′ and 5′-CTAGCTCCGGCGCCCAATCACCGCGGG-3′ and bim CCAAT mut 5′-CTAGCCCGCGGTGAGCTCGCGCGGAG-3′ and 5′-CTAGCTCCGGCGCGAGCTCACCGCGGG-3′ (the mutated bases of the ICB are underlined). Oligonucleotides containing a CCAAT box from the rat GTP C1 gene were used as a positive control: GTP C1 wt 5′-CTAGGCTCGGCCAATGAGAACGCCT-3′ and 5′-CTAGAGGCGTTCTCATTGGCCCGAGC-3′ and GTP C1 mut 5′-CTAGCTCGGGGAGCTGAGAACGCCT-3′ and 5′-CTAGAGGCGTTCTCAGCTCCCCGAGC-3′.¹² Binding reactions were set up in a 20 μl volume as described previously,¹² with 5 μg of nuclear extract from PC6-3 cells and 10 μg of whole cell extract from sympathetic neurons. Supershift experiments were carried out by incubating samples on ice with antibodies (4 μg) for 2 h. The Santa Cruz NF-YA (H-209:Sc-10779 X) antibody was used in EMSA experiments along with c-Jun (H-79:Sc-1694 X) or Fra-2 (L-15:Sc-171 X) antibodies as negative controls (Santa Cruz). Samples were incubated with 0.4 ng of ³²P-labelled oligonucleotide for 15 min at room temperature and run on a 5% polyacrylamide gel. After electrophoresis, gels were fixed in 10% acetic acid/10% methanol, dried and bands were visualised by exposing the gel to MXB X-ray film (Kodak, Rochester, NY, USA).

Chromatin immunoprecipitation

Differentiated PC6-3 cells (∼4 × 10⁶ cells per 9 cm dish) were either maintained in the presence of NGF, or withdrawn from NGF for 16 h. The cells were fixed in 1% formaldehyde for 3 h at room temperature and ChIP was performed using a ChIP assay kit from Upstate (Millipore) with some modifications, as described previously.¹⁹ The following antibodies from Santa Cruz were used for ChIP: NF-YA (H-209:Sc-10779 X), p300 (C-20:Sc-585 X), CBP (C-20:Sc-583 X) and FOXO3a (H-144:Sc-11351 X). Millipore Bim (AB17003) and Santa Cruz MyoD (M-318:Sc-760 X) antibodies were used as negative controls.

ChIP samples were analysed by semi-quantitative PCR. To detect binding of NF-Y to the ICB in the rat bim promoter the following primer pair was used: 5′-CAGGCCAAGTCACTAGGGTAAACAC-3′ and 5′-GAATCCGGTGACTGCAACGACAA-3′. The same primer pair was used to detect binding of p300 and CBP to this region. The following primer pairs were used to detect binding of FOXO3a to FOXO site 1: 5′-CTAAGTTCCGCTCTGCGAGGT-3′ and 5′-TGCAGGCTGCGACAGGTAGT-3′, and FOXO3a to FOXO site 2: 5′-GATTCACACCACCTCCGCT-3′ and 5′-GCCTGTCTTCTATCCATCCC-3′. Samples were analysed between 25 and 30 cycles and the average amount of bim promoter DNA, precipitated by each antibody, was calculated using ImageQuant image analysis software (GE Healthcare, Waukesha, WI, USA).

Co-immunoprecipitation

The co-immunoprecipitation (co-IP) experiments were performed on nuclear lysates from differentiated PC6-3 cells maintained in +NGF medium or withdrawn from NGF for 16 h. A 50% slurry of protein A/G-agarose (Santa Cruz) was prepared in PBS (−) and added to a 1.5-ml microfuge tube containing 2 μg of antibody to immunoprecipitate either FOXO3a (Cell Signaling; #2497), p300, CBP or NF-Y (NF-Y, p300 and CBP antibodies were the same for co-IP and ChIP), and resuspended in 500 μl of ice-cold PBS (−). This was also repeated for the non-specific control, purified rabbit immunoglobulin in PBS (Jackson ImmunoResearch). The mixtures were incubated at 4°C for 90 s and the beads were collected by centrifugation at 15 700 × g for 2 s at 4°C. The supernatant containing the unbound antibody was removed and the beads were resuspended in 1 ml of non-denaturing lysis buffer (1% v/v Triton-X100, 50 mM Tris-HCl pH 7.4, 300 mM NaCl, 5 mM EDTA, 0.02% v/v NaN₃ pH 7.4, 1 mM PMSF)_. The tubes were inverted four times to mix and the beads were collected by centrifugation as previously. The beads were then washed in 1 ml of non-denaturing lysis buffer and collected by centrifugation. Around 80 μg of nuclear lysate was added to each tube containing specific or control antibody-bound beads. The mixture was incubated at 4°C for 2 h with rotation. The beads were then collected by centrifugation and the supernatant was removed. The beads were resuspended in 1 ml of ice-cold wash buffer, the tubes inverted four times to mix, and the beads were collected by centrifugation as previously. This wash step was repeated three times and the final supernatant was removed to leave the beads in ∼15 μl of wash buffer (containing the immune complexes). The beads were resuspended in 30 μl of SDS gel sample buffer, boiled for 5 min at 100°C and analysed by immunoblotting as described previously.⁷ The NF-YA, FOXO3a, p300 and CBP antibodies used for immunoprecipitation were also used for immunoblots and an ERK1/2 antibody (Cell Signaling; # 9102) was used to check protein loading.

To prepare nuclear extracts for co-IP, neuronally differentiated PC6-3 cells were washed twice in ice-cold PBS (−) and then scraped off the dishes in 2 ml of PBS (per 9 cm dish). Cells were pelleted by centrifugation at 400 × g for 5 min at 4°C and resuspended in 200 μl of buffer A (10 mM Hepes pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF) before incubation on ice for 15 min. NP40 (10%) was added to each lysate (to a final concentration of 1%) and the samples were vortexed for 15 s before incubation for 15 min at 4°C. The lysates were pelleted by centrifugation at 13 000 × g for 30 s, resuspended in 200 μl of ice-cold buffer C (20 mM Hepes pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 25% v/v glycerol) and incubated at 4°C with shaking. After 1 h, lysates were vortexed for 30 s and pelleted by centrifugation for 10 min at 15 700 × g at 4°C. The supernatant was removed and this was the nuclear extract.

Statistical analysis

In each set of experiments, data are normalised to a control sample (for example, bim-LUC +NGF is set to one). For microinjection, the relative induction of a DNA construct is calculated by dividing the normalised luciferase activity in the absence of NGF by the normalised luciferase activity in the presence of NGF. All data are presented as the mean±S.E. of multiple experiments.

The statistical significance of differences between means was evaluated by performing an unpaired Student's t-test (for two-tailed distributions), followed by a Bonferroni correction. To compare normalised data with the control sample, which has no error associated to it (for example, when comparing data to bim-LUC +NGF, which is set to one), the log10 values of the data were taken and a one sample t-test was used (for two-tailed distributions). Statistical significance is presented as P-values in the Figures.

Abbreviations

NGF:: nerve growth factor
ICB:: inverted CCAAT box
EMSA:: electrophoretic mobility shift assay
ChIP:: chromatin immunoprecipitation
GTP C1:: GTP cyclohydrolase I
co-IP:: co-immunoprecipitation
ss:: supershift
gp IgG:: guinea pig IgG
TRD:: Texas red dextran
CGN:: cerebellar granule neuron

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Acknowledgements

We thank Tony Kouzarides for providing the expression vector for CBP, Becky Randall for advice on EMSA assays, and Francesca Menghi and Mike Hubank for critical reading of the manuscript. This work was supported by The Wellcome Trust (Senior Research Fellowship in Basic Biomedical Science 057700 to JH). RM is supported by the Associazione Italiana Ricerca Cancro (AIRC). SD and IL are supported by the Centre National de la Recherche Scientifique (CNSR) and the Institut National de la Santé et de la Recherche Médicale (INSERM).

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Authors and Affiliations

Molecular Haematology and Cancer Biology Unit, Institute of Child Health, University College London, London, UK
R Hughes, M Kristiansen & J Ham
Institut de Génétique Moléculaire de Montpellier,
I Lassot & S Desagher
Université Montpellier 1, Université Montpellier 2, Montpellier, France
I Lassot & S Desagher
Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Milan, Italy
R Mantovani

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R Hughes
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M Kristiansen
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I Lassot
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S Desagher
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R Mantovani
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Correspondence to J Ham.

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Edited by L Greene

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Hughes, R., Kristiansen, M., Lassot, I. et al. NF-Y is essential for expression of the proapoptotic bim gene in sympathetic neurons. Cell Death Differ 18, 937–947 (2011). https://doi.org/10.1038/cdd.2010.166

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Received: 12 May 2010
Revised: 18 October 2010
Accepted: 12 November 2010
Published: 17 December 2010
Issue Date: June 2011
DOI: https://doi.org/10.1038/cdd.2010.166

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