Sp3/REST/HDAC1/HDAC2 Complex Represses and Sp1/HIF-1/p300 Complex Activates ncx1 Gene Transcription, in Brain Ischemia and in Ischemic Brain Preconditioning, by Epigenetic Mechanism

Abstract

The Na(+)-Ca(2+) exchanger 1 (NCX1) is reduced in stroke by the RE1-silencing transcription factor (REST), whereas it is increased in ischemic brain preconditioning (PC) by hypoxia-inducible factor 1 (HIF-1). Because ncx1 brain promoter (ncx1-Br) has five putative consensus sequences, named Sp1A-E, for the specificity protein (Sp) family of transcription factors (Sp1-4), we investigated the role of this family in regulating ncx1 transcription in rat cortical neurons. Here we found that Sp1 is a transcriptional activator, whereas Sp3 is a transcriptional repressor of ncx1, and that both bind ncx1-Br in a sequence-specific manner, modulating ncx1 transcription through the Sp1 sites C-E. Furthermore, by transient middle cerebral artery occlusion (tMCAO) in rats, the transcriptional repressors Sp3 and REST colocalized with the two histone-deacetylases (HDACs) HDAC1 and HDAC2 on the ncx1-Br, with a consequent hypoacetylation. Contrarily, in PC+tMCAO the transcriptional activators Sp1 and HIF-1 colocalized with histone acetyltransferase p300 on ncx1-Br with a consequent hyperacetylation. In addition, in neurons silenced with siRNA of NCX1 and subjected to oxygen and glucose deprivation (OGD) (3 h) plus reoxygenation (RX) (24 h), the neuroprotection of Class I HDAC inhibitor MS-275 was counteracted, whereas in neurons overexpressing NCX1 and subjected to ischemic preconditioning (PC+OGD/RX), the neurotoxic effect of p300 inhibitor C646 was prevented. Collectively, these results demonstrate that NCX1 expression is regulated by the Sp3/REST/HDAC1/HDAC2 complex in tMCAO and by the Sp1/HIF-1/p300 complex in PC+tMCAO and that epigenetic intervention, by modulating the acetylation of ncx1-Br, may be a strategy for the development of innovative therapeutic intervention in stroke.

Keywords: REST\NRSF; acetylation; brain ischemia; epigenetic; ischemic brain preconditioning; sodium/calcium exchanger.

Figure 1.

Transcription factors Sp1 and Sp3, but not Sp4, bind to ncx1 brain promoter sequence from −366 to 58. a, Map of the rat ncx1 gene indicating location of Sp1, HIF-1 (HRE), REST (RE-1) sequences contained in distal [ncx1-Br (I)] and proximal [ncx1-Br (II)] regions of ncx1 brain promoter and of PCR primers used to detect the presence of specific DNA sequences in ChIP complexes. Triangles represent the 5 Sp1 identified putative motifs in ncx1-Br, named Sp1A–E. Rectangles and square represent the 2 HRE sites and RE-1 site, respectively. b, ChIP analysis of the ncx1-Br (I) and ncx1-Br B (II) regions performed with anti-Sp1, anti-Sp3, and anti-Sp4. Anti-IgG was used as negative control. The binding activity of Sp1, Sp3, and Sp4 is graphically represented as the percentage of total input of chromatin DNA. c, ChIP analysis of the NT3 promoter region performed with anti-Sp4. Anti-IgG was used as negative control. The binding activity of Sp4 is graphically represented as the percentage of total input of chromatin DNA. *p < 0.05 versus IgG. Each column represents the mean ± SEM (n = 3).

Figure 2.

Cortical neurons transfected with siRNAs or constructs for Sp1, Sp3, and Sp4. a–c, Representative WB and quantification of Sp1, Sp3, and Sp4 protein expression in neurons after treatment with siCTL, siSp1, siSp3, and siSp4. d–f, Representative WB and quantification of Sp1, Sp3, and Sp4 protein expression in neurons after transfection with empty vector (pN3), and after Sp1, Sp3, and Sp4 overexpression. Each column represents the mean ± SEM (n = 3). *p < 0.05 versus respective siCTL or pN3.

Figure 3.

Sp1 is a transcriptional activator, whereas Sp3 is a transcriptional repressor of ncx1 in cortical neurons. a–c, Luciferase assay of ncx1-Br, qRT-PCR, and representative WB with quantification of NCX1 under control conditions (CTL) and after treatment with siCTL, siSp1, siSp3, siSp4, and in combination with siSp1 and siSp3 (n = 4). Each column represents the mean ± SEM. *p < 0.05 versus pGL3 ncx1, siCTL. d–f, Luciferase assay of ncx1-Br, qRT-PCR, and representative WB with quantification of NCX1 under control conditions (CTL) and after transfection with pN3 or overexpression of Sp1, Sp3, Sp4, and after combination of Sp1 and Sp3 (n = 3). Each column represents the mean ± SEM. *p < 0.05 versus pGL3 ncx1, pN3.

Figure 4.

Effect of Sp1 and Sp3 on the transcriptional activity of ncx1-Br after site-directed mutagenesis of the putative Sp1 sites and analysis of their binding on ncx1-Br sequence. a, The core consensus sequences of the five putative Sp1A–E sites are boxed, and the locations relative to the reported transcription start site are separately numbered. Mutated nucleotides of the particular sequences are underlined, and the replaced nucleotides are shown in italics below the wild-type sequences. The bent arrows indicate the reported transcription start site. b, c, Cortical neurons transiently overexpressing Sp1 or Sp3 were cotransfected with pGL3-ncx1 or pGL3-ncx1/Sp1Bmut, pGL3-ncx1/Sp1Cmut, pGL3-ncx1/Sp1Dmut, pGL3-ncx1/Sp1Emut, and pGL3-ncx1/Sp1CDEmut constructs. Twenty-four hours after transfection, neurons were lysed in 1× passive lysis buffer. Lysates were analyzed for luciferase activity. Luciferase activity was expressed as firefly-to-renilla ratio. Each column represents the mean ± SEM (n = 4). *p < 0.05 vs pGL3-ncx1. **p < 0.05 versus all. d, EMSA was performed incubating Cy5′-Sp1 consensus sequence probe without or with nuclear extracts from SH-SY5Y cells (CTL NEG and CTL, respectively, lanes 1 and 2). Competition experiments were performed with increasing molar excess (20- and 100-fold) of the unlabeled wild-type ncx1/Sp1B–E (ncx1/Sp1B, lanes 3 and 4; ncx1/Sp1C, lanes 7 and 8; ncx1/Sp1D, lanes 11 and 12; ncx1/Sp1E, lanes 15 and 16) and the mutant ncx1/Sp1B–E probes (ncx1/Sp1B, lanes 5 and 6; ncx1/Sp1C, lanes 9 and 10; ncx1/Sp1D, lanes 13 and 14; ncx1/Sp1E, lanes 17 and 18). Antibodies specific for Sp1 and Sp3 were added to the EMSA reaction as indicated (lanes 19 and 20). e–g, Nuclear extracts from SH-SY5Y cells were incubated with the Cy5′-tagged ncx1-Sp1C–E (CTL, lanes 1). Competition experiments were performed with increasing molar excess (100-fold) of the unlabeled wild-type ncx1/Sp1C–E (ncx1/Sp1C–E, lanes 2) and the mutant ncx1/Sp1C–E (ncx1/Sp1C–E, lanes 3). Nuclear extracts were incubated in the presence of anti-Sp1, anti-Sp3, and anti-Sp4 (lanes 4–6). h, i, Chromatin was prepared from SHSY-5Y cells transiently transfected with Sp1, Sp3, and pN3 (empty vector) expressing plasmids and with the pGL3-ncx1 or pGL3-ncx1/Sp1Cmut, pGL3-ncx1/Sp1Dmut, pGL3-ncx1/Sp1Emut, and pGL3-ncx1/Sp1CDEmut constructs. Chromatin from transfected cells was immunoprecipitated with Sp1 and Sp3 antibodies. Exogenous DNA containing either the ncx1-Br fragment (−340/151, pGL3-ncx1) or the mutated Sp1C–E sequence alone or in combination (pGL3-ncx1/Sp1Cmut, pGL3-ncx1/Sp1Dmut, pGL3-ncx1/Sp1Emut, and pGL3-ncx1/Sp1CDEmut constructs) was amplified using ncx1-Br forward II promoter-specific forward primer and a luciferase gene-specific reverse primer (LucNrev primer). The binding activity of Sp1 is graphically represented as the percentage of total input of chromatin DNA. *p < 0.05 versus cells cotransfected with pN3 and pGL3-ncx1. **p < 0.05 versus cells cotransfected with constructs overexpressing Sp1 or Sp3 and pGL3-ncx1. ^#p < 0.05 versus all. Each column represents the mean ± SEM (n = 3).

Figure 5.

Effect of tMCAO and PC+tMCAO on NCX1, HIF-1, REST, Sp1, Sp3, and Sp4 in the rat ipsilateral–temporoparietal cortex. a–f, Representative WB with quantification of NCX1, HIF-1, REST, Sp1, Sp3, and Sp4 protein expression in: (1) control group (CTL), (2) PC, (3) tMCAO, and (4) PC+tMCAO. Each column represents the mean ± SEM (n = 5 animals for each column). *p < 0.05 versus CTL. **p < 0.05 versus all. ^#p < 0.05 versus tMCAO.

Figure 6.

tMCAO and PC+tMCAO rat models promote REST/Sp3/HDAC1/HDAC2 and HIF-1/Sp1/p300 colocalization on ncx1-Br in the ipsilateral–temporoparietal cortex, respectively. a–d, ChIP analysis of ncx1-Br in the ipsilateral–temporoparietal cortex in (1) CTL, (2) PC, (3) tMCAO, and (4) PC+tMCAO. The binding activity of HIF-1, REST, Sp1, and Sp3 is graphically represented as the percentage of total input of chromatin DNA. Anti-IgG was used as negative control. Each column represents the mean ± SEM (n = 6 animals for each column). *p < 0.05 versus CTL. e–i, Re-ChIP analysis of ncx1-Br in the ipsilateral–temporoparietal cortex in (1) CTL, (2) PC, (3) tMCAO, and (4) PC+tMCAO. Primary ChIP products for anti-Sp3 (I Ab) were subjected to re-ChIP with anti-REST, anti-HDAC1, and anti-HDAC2 (II Ab) (e, g, h) but for anti-Sp1 (I Ab) were subjected to re-ChIP with anti-p300 (II Ab) (f, i). Anti-IgG was used as negative control. The input DNA lane represents 5% of the precleared chromatin used in each ChIP reaction. The figure is representative of two independent experiments.

Figure 7.

Effect of tMCAO and PC+tMCAO rat models on H3 acetylation, RNA-Pol II, HDAC1, HDAC2, and p300 binding to ncx1-Br. a–e, ChIP analysis of ncx1-Br in the ipsilateral–temporoparietal cortex in (1) CTL, (2) PC, (3) tMCAO, and (4) PC+tMCAO. The binding activity of H3 acetyl, RNA-Pol II, HDAC1, HDAC2, and p300 is graphically represented as the percentage of total input of chromatin DNA. IgG was used as negative control. Each column represents the mean ± SEM (n = 6 animals for each column). *p < 0.05 versus CTL. **p < 0.05 versus all.

Figure 8.

Sp1, Sp3, HDAC1, HDAC2, and p300 protein expression in rat brain after siRNA treatment. a–e, Representative Western blots of Sp3, HDAC1, HDAC2, Sp1, and P300 protein levels in the temporoparietal cortex of rats intracerebroventricularly treated with siSp3, siHDAC1, siHDAC2, siSp1, and siP300. Each column represents the mean ± SEM (n = 3). *p < 0.05 versus respective siCTL.

Figure 9.

NCX1 is epigenetically modulated by Sp3/REST/HDAC1/2 complex in tMCAO and by Sp1/HIF-1/p300 in PC+tMCAO. a, ChIP analysis with anti-H3 acetyl of ncx1-Br in CTL and in tMCAO after intracerebroventricular injection of siCTL, siSp3, siREST, siREST+siSp3, and siHDAC1+siHDAC2. IgG was used as negative control. Each column represents the mean ± SEM (n = 5 animals for each column). *p < 0.05 versus CTL. **p < 0.05 versus all. b, c, qRT-PCR and representative WB with quantification of NCX1 in CTL and in tMCAO after intracerebroventricular injection of siCTL, siSp3, siREST, siREST+siSp3, and siHDAC1+siHDAC2. Each column represents the mean ± SEM (n = 6 animals for each column). *p < 0.05 versus CTL. **p < 0.05 versus all. d, ChIP analysis with anti-Sp3 (black bars) and anti-REST (gray bars) of ncx1-Br in CTL and in tMCAO after intracerebroventricular injection of siCTL, siSp3, siREST, siREST+siSp3, and siHDAC1+siHDAC2. IgG was used as negative control. Each column represents the mean ± SEM (n = 6 animals for each column). *,^p < 0.05 versus CTL. *,^^p < 0.05 versus all. e, ChIP analysis with an anti-H3 acetyl of ncx1-Br in CTL and in PC+tMCAO after intracerebroventricular injection of siCTL, siSp1, siHIF-1, siHIF-1+siSp1, and sip300. IgG was used as negative control. Each column represents the mean ± SEM (n = 6 animals for each column). *p < 0.05 versus CTL. f, g, qRT-PCR and representative WB with quantification of NCX1 in CTL and PC+tMCAO after intracerebroventricular injection of siCTL, siSp1, siHIF-1, siHIF-1+siSp1, and sip300. Each column represents the mean ± SEM (n = 6 animals for each column). *p < 0.05 versus CTL. h, ChIP analysis with anti-Sp1 (black bars) and anti-HIF-1 (gray bars) of ncx1-Br in CTL and in PC+tMCAO after intracerebroventricular injection of siCTL, siSp1, siHIF-1, and siHIF-1+siSp1. IgG was used as negative control. Each column represents the mean ± SEM (n = 5 animals for each column). *,^p < 0.05 versus CTL. *,^^p < 0.05 versus all.

Figure 10.

NCX3 is not modulated by the Sp3/REST/HDAC1/2 complex in tMCAO and by Sp1/HIF-1/p300 in PC+tMCAO. a, b, qRT-PCR and representative WB with quantification of NCX3 in CTL and in tMCAO after intracerebroventricular injection of siCTL, siSp3, siREST, siREST+siSp3, and siHDAC1+siHDAC2. Each column represents the mean ± SEM (n = 5 animals for each column). *p < 0.05 versus CTL. **p < 0.05 versus all. c, d, qRT-PCR and representative WB with quantification of NCX3 in CTL and PC+tMCAO after intracerebroventricular injection of siCTL, siSp1, siHIF-1, siSp1+siHIF-1, and sip300. Each column represents the mean ± SEM (n = 5 animals for each column). *p < 0.05 versus CTL.

Figure 11.

Effect of Class I HDAC inhibitor MS-275 and of HAT p300 inhibitor C646 on cell survival and NCX1 protein expression in cortical neurons exposed to OGD/RX or to PC+OGD/RX, respectively. a, Representative WB with quantification of H3 acetyl in neurons subjected to OGD/RX alone or with MS-275. Each column represents the mean ± SEM (n = 3). *p < 0.05 versus OGD/Rx. b, LDH assay in (1) control condition (CTL), (2) OGD/RX+vehicle, (3) OGD/RX+siCTL, (4) OGD/RX+MS-275 (1 μm), (5) OGD/RX+MS-275+siNCX1, and (6) OGD/RX+siNCX1. Each column represents the mean ± SEM (n = 5). *p < 0.05 versus CTL. **p < 0.05 versus OGD/Rx+vehicle and OGD/Rx+siCTL. ^#p < 0.05 versus OGD/Rx+MS-275. ***p < 0.05 versus all. c, NCX1 protein expression with quantification in (1) control condition (CTL), (2) OGD/RX, (3) OGD/RX+vehicle, (4) OGD/RX+siCTL, (5) OGD/RX+MS-275 (1 μm), and (6) OGD/RX+MS-275+siNCX1. Each column represents the mean ± SEM (n = 3). *p < 0.05 versus all. d, Representative WB with quantification of H3 acetyl in neurons subjected to PC+OGD/RX alone or with C646. Each column represents the mean ± SEM (n = 3). *p < 0.05 versus OGD/Rx. e, f, LDH assay and NCX1 protein expression with quantification in (1) CTL, (2) PC, (3) OGD/RX, (4) PC+OGD/RX, (5) PC+OGD/RX+vehicle, (6) PC+OGD/RX+empty vector, (7) PC+OGD/RX+C646, and (8) PC+OGD/RX+C646+NCX1. Each column represents the mean ± SEM (n = 3 for LDH assay and for WB). *p < 0.05 versus CTL. **p < 0.05 versus all. ^#p < 0.05 versus PC+OGD/RX alone or with vehicle and empty vector. ^p < 0.05 versus PC+OGD/RX+C646.