1. Volume 21, Issue 2, Pages 283-294 (January 2006)
A Nitric Oxide Signaling Pathway Controls CREB-Mediated Gene Expression in Neurons 
Antonella Riccio, Rebecca S. Alvania, Bonnie E. Lonze, Narendrakumar Ramanan, Taeho Kim, Yunfei Huang, Ted M. Dawson, Solomon H. Snyder, David D. Ginty 
Molecular Cell 
Volume 21, Issue 2, Pages (January 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1
BDNF-Dependent CREB Binding to Endogenous CRE Sequences in Cortical Neurons
(A) ChIP analysis of cortical neurons treated with BDNF (75 ng/ml) for the indicated times. ChIPs performed with anti-CREB antibody were followed by PCR amplification of the c-fos (top), nNOS (middle), and VGF (bottom) promoter regions containing their CREs. Immunoprecipitation input controls contain c-fos promoter sequences detectable by PCR (bottom) (n = 4). Although there is variability in the temporal pattern of CREB DNA binding, in all cases an increase in CREB binding occurred within 5 min of neuronal stimulation.
(B) ChIP assays were performed with anti-CREB, and levels of immunoprecipitated c-fos, nNOS, and VGF promoters were measured by quantitative PCR. Data are represented as fold changes over unstimulated cortical neurons (n = 4–6). Shown are the means ± SEM.
(C) ChIP analysis of BDNF-treated cortical neurons derived from wild-type (left panels) or Creb null (right panels) mouse embryos. Neurons were stimulated with BDNF (75 ng/ml) for the indicated times. Immunoprecipitations were performed with anti-CREB (top) or anti-CBP (middle). PCR amplification of the c-fos promoter CRE indicates association of CREB and CBP with this DNA region (n = 2).
(D) ChIP analysis of cortical neurons stimulated with BDNF (75 ng/ml) and immunoprecipitated with CREB (Upstate), CREB (Cell Signaling), SRF, or acetyl histone H4 antibodies followed by PCR amplification for c-fos (n = 3).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 CREB Binding to DNA Is Stimulus and Cell Type Specific
(A) ChIP analysis of cortical neurons maintained in cultures for 20 days and stimulated with bicuculline (50 μM) for the indicated times. CREB immunoprecipitation and PCR for c-fos (n = 3).
(B) ChIP analysis of primary fibroblasts treated with EGF (30 ng/ml) as indicated. CREB immunoprecipitation and PCR for c-fos (top), nNOS (middle), and VGF (bottom) promoters are shown (n = 3). Abbreviation: PI, preimmume antibody.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3
BDNF Induces CREB Phosphorylation and CREB DNA Binding through Independent Signaling Pathways
(A) ChIP analysis of BDNF-stimulated cortical neurons in the presence (right panels) or the absence (left panels) of the MEK inhibitor UO-126. Neurons were pretreated with UO-126 (30 μM) for 1 hr prior to stimulation. CREB and acetyl histone H4 immunoprecipitation followed by PCR revealed association of CREB with the c-fos promoter (upper panels) under both conditions (n = 4).
(B) Immunoblot analysis of phospho-CREB (top panels), phospho-ERK (middle panels), and CREB (bottom) in lysates from neurons subjected to the same treatment conditions described in (A) (n = 3).
(C) ChIP analysis of BDNF-treated neurons in the presence (right lanes) or absence (left lanes) of the calcium chelator BAPTA-AM (50 μM). ChIPs were performed with anti-CREB, anti-CBP, and anti-acetyl histone H4. PCR analysis of the c-fos gene revealed an inhibition of CREB and CBP binding and histone H4 acetylation (n = 3).
(D) Immunoblot analysis of phospho-CREB (top) and CREB (bottom) in lysates from neurons subjected to the same treatment conditions described in (C).
(E) Real-time PCR quantitation of ChIP analyses of cortical neurons treated with BDNF (75 ng/ml) for the indicated times after pretreatment with either vehicle control, the MEK inhibitor U0-126 (30 μM), or BAPTA-AM (BAPTA, 50 μM). Data are represented as fold changes over unstimulated cortical neurons (n = 3, ∗p < 0.01). Shown are the averages ± SEM.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
Stimulus-Dependent Increase in CREB DNA Binding Occurs Independently of Phosphorylation of CREB Ser133
(A) Stimulus-inducible CREB-DNA binding occurs independently of Ser-133. ChIP and immunoblot analyses of PC12 cells and HEK293 cells (B) treated with NGF (50 ng/ml) or forskolin (10 μM), respectively, for the indicated times. Cells were transiently transfected with the myc-tagged CREB constructs indicated. ChIP assays were carried out with an anti-myc antibody to detect the DNA binding activity of the transfected myc-CREB fusion proteins (n = 4). Abbreviation: WB, immunoblot.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 NO Signaling Regulates CREB-DNA Binding
(A) BDNF induces a rapid increase of NOS activity. Cortical neurons were either pretreated with the NOS inhibitor L-NMMA (500 μM) or UO-126 (30 μM) for 1 hour prior to stimulation or left untreated. Cells were stimulated with PBS or BDNF (75 ng/ml) for 30 min, as indicated. HEK 293 cells overexpressing nNOS were stimulated with ionomycin (5 μM). NOS activity was determined by assessing the conversion of [3H] arginine to [3H] citrulline (n = 3, ∗p < 0.05, ∗∗p < 0.001). Shown are the means ± SEM.
(B) ChIP analysis of cortical neurons stimulated with BDNF as indicated either in the presence (right panels) or absence (left panels) of L-NAME (2 mM). CREB and acetyl histone H4 immunoprecipitation was followed by PCR amplification of the c-fos (top panels) and nNOS (bottom panels) CREs (n = 5).
(C) Immunoblot analysis of phospho-CREB, acetyl histone H4, and tubulin in lysates from neurons subjected to the same treatment conditions described in (B) (n = 3).
(D) Real-time PCR quantitation of ChIP analyses of cortical neurons treated with BDNF (75 ng/ml) for the indicated times after pretreatment with either vehicle control, L-NAME (2 mM), or NO donors (SNAP and NOC-12, 100 μM each). Data are represented as fold changes over unstimulated cortical neurons (n = 4, p < 0.01). Shown are the means ± SEM.
(E) ChIP analysis of cortical neurons stimulated with BDNF (75 ng/ml) as indicated in the presence of or absence of NO donors, N-Ethyl-2 ethanamine-12 (NOC-12, 100 μM), and S-Nitroso-N-acetylpenicillamine (SNAP, 100 μM). CREB and acetyl histone H4 immunoprecipitations were followed by PCR for the c-fos promoter (n = 3).
(F) Phospho-CREB and total CREB immunoblot analysis of cortical neuron lysates that had been treated as described in (E) (n = 3). The small change in CREB phosphorylation in SNAP and NOC-12-treated cells was not consistently observed.
(G) ChIP analysis of BDNF-treated cortical neurons derived from wt (left lanes) or nNOS null (right lanes) mouse embryos. Neurons were stimulated with BDNF (75 ng/ml) for the indicated times. Immunoprecipitations were performed with an acetyl histone H4 antibody followed by real-time PCR for the c-fos promoter (n = 2).
(H) Phospho-CREB and CREB immunoblot analysis of cortical neuron obtained from wt (left panels) or nNOS null mice (right panels) treated with BDNF (75 ng/ml) for the indicated times (n = 2).
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 6 NO Influences the Expression of CREB Target Genes
(A and B) Northern blot analysis of neurons treated simultaneously with BDNF and the NO donor SNAP. Neurons were stimulated with the indicated concentrations of SNAP in the presence or absence of BDNF (1 ng/ml) for 45 min (n = 3). Treatment with increasing concentrations of SNP (B) yielded similar results (n = 3).
(C) Northern blot analysis of cortical neurons stimulated with BDNF for the indicated times either in the presence or absence of L-NMMA (500 μM). Cells were pretreated with L-NMMA or PBS for 30 min and stimulated with BDNF (75 ng/ml) for the times indicated (n = 4). Neurons pretreated with L-NAME (2 mM) yielded similar results.
(D) Expression of c-fos in brain of mice lacking nNOS. In situ hybridization of sagittal sections of cortex obtained from 10–12-week-old mice either maintained in standard cages (top panels) or exposed to novel enriched environment (NEE) (bottom panels) for 45 min (N = 3). Exposure to NEE greatly enhances c-fos expression in cortical neurons. Note that the absence of nNOS results in a decrease in NEE-induced c-fos expression.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 Nitrosylation of Proteins Associated with the c-fos CRE
(A) CREB DNA binding is independent of cGMP/PKG signaling. Neurons were pretreated with DT3 (100 nM) for 60 min and treated with BDNF (75 ng/ml) for the indicated times. Immunoprecipitation of CREB and acetyl histone H4 were followed by PCR of the c-fos promoter (n = 3).
(B) Immunoblot analysis of phospho-CREB, and CREB in lysates from neurons subjected to the same treatment conditions described in (A) (n = 3).
(C) ChIP analysis of cortical neurons stimulated with BDNF (75 ng/ml) as indicated (lanes 1–4) or pretreated with either NOS inhibitors (L-NAME, 2 mM or L-NMMA, 500 μM) or NO donors (SNAP and NOC-12, 100 μM each) (lanes 5–7). Antinitro-cysteine immunoprecipitation of cell lysates were followed by PCR analysis of the c-fos promoter (n = 3).
(D) NO regulates the binding of HDAC2 to the c-fos NO regulates the binding of HDAC2 to the c-fos promoter. Cortical neurons were stimulated with BDNF in the absence (lanes 1–4) or presence (lanes 5 and 6) of NOS inhibitors (L-NAME, 2 mM and L-NMMA, 500 μM). HDAC2 was immunoprecipitated, and PCR for c-fos was performed. NO donors were added to unstimulated cells (lane 7) (n = 2).
(E) Expression of HDAC2 in cortical neurons treated with BDNF (75 ng/ml) for the indicated times in the absence (lanes 1–4) or presence (lane 5) of L-NAME (2 mM) (n = 2).
(F) Inhibition of histone deacetylases induces CREB DNA binding. ChIP analysis of cortical neurons treated with trichostatin A at the indicated concentrations and immunoprecipitated with CREB and acetyl-histone H4 antibodies is shown. PCR of c-fos promoter is shown (n = 3).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions