Identification of a CRE/AP-1-like motif and GATA-binding site within the Syncytin-2 promoter.

<p><b>(A)</b> Extended probe 5 (termed WT) (from-211 to-177) (indicated in the upper part of the panel) was used for EMSA analyses and highlights the CRE/AP-1-like motif and the potential GATA-binding site in the Syncytin-2 promoter. Nuclear extracts from BeWo cells treated (T) or not (NT) with forskolin for 15 min were pre-incubated with different concentrations of cold specific or non-specific oligonucleotides prior to addition of the labeled WT probe. (<b>B</b>) EMSA analyses of nuclear extracts from BeWo cells stimulated with forskolin at different time points and incubated with the WT probe. <b>(C)</b> Representation of the various oligonucleotides bearing mutated sequences (nucleotides in italic) used in EMSA analyses. <b>(D-E)</b> Nuclear extracts from non-treated (NT) or forskolin-treated (T) BeWo cells were incubated with the WT probe and excess (100X) cold WT or mutated oligonucleotides presented in (<b>C</b>). Specific DNA-protein complexes are indicated on left side of panels. (<b>F</b>) Nuclear extracts from primary villous cytotrophoblasts cultured for 24 and 96 hours were incubated with the WT probe and with or without excess (100X) cold WT oligonucleotide.</p

CREB2 and JunD overexpression induce BeWo fusion.

<p>BeWo cells were either left untreated (Control) or stimulated with forskolin or SP-cAMP (vs. its control, RP-cAMP) (<b>C-D</b>). In parallel, BeWo cells were transfected with expression vectors for JunD or CREB2 (or corresponding empty vectors) (200 ng). At 48 h post-transfection or post-stimulation, cell fusion was analyzed with anti-desmoplakin antibodies for membrane staining (green) and propidium iodide for nuclei staining (red). Images were visualized by confocal microscopy. <b>(B, D)</b> Cell fusion index was calculated in stimulated or transfected cells as follow: a total of 200 nuclei were counted in five independent fields per condition (average total of 1000 nuclei) and a percentage was calculated for the number of nuclei comprised in syncytia. These results are representative of a three different experiments (*p< 0.05; ***p< 0.001).</p

Analysis of the-300 to-150 Syncytin-2 region responsive to forskolin.

<p><b>(A)</b> Sequence of the-300 to-150 promoter region of Syncytin-2 in which putative transcription binding sites are underlined. <b>(B-C)</b> The pGL2-TATA-150 vector comprising the-300 to-150 Syncytin-2 promoter region and a TATA box upstream of the luciferase reporter gene (vs. the pGL2-TATA control vector) (400 ng) was co-transfected along with 200 ng of pRcActin-LacZ in BeWo <b>(B)</b> or 293T cells <b>(C)</b>. After transfection, cells were either left untreated (Control) or treated with 50 μM of forskolin for 12 h. Cells were harvested and analyzed for relative luciferase expression. Luciferase activity values were normalized for β-galactosidase activity and are expressed as the mean normalized RLU ± S.E.M. from three independently transfected cells in the same experiment. These results are representative of three independent experiments (*p< 0.05; *** p< 0.001). <b>(D)</b> Five different probes covering the-300 to-150 region were used for EMSA. Nuclear extracts (10 μg) from non-treated (NT) or treated (T) BeWo cells were incubated with the different labeled probes covering this 150 nt region. The arrow points to a specific signal observed with probe 5.</p

CREB2 induces activation of the Syncytin-2 promoter.

<p><b>(A)</b> BeWo cells were co-transfected with pGL3/2600 (200 ng) (or pGL3basic), KCREB, a CREB dominant-negative mutant expression vector (vs. control vector) (200 ng) and pRcActin-LacZ (200 ng). At 36 h post transfection, cells were either left untreated (Control) or treated with forskolin. After 12 h of treatment, cells were harvested and analyzed for luciferase activity in triplicates. Luciferase activities were normalized over β-galactosidase activity and are expressed as the mean normalized RLU ± S.E.M. from three independently transfected cells in the same experiment. Results are representative of three independent experiments. <b>(B)</b> BeWo cells were co-transfected with pGL3/600 (200 ng), the constitutively activated CREB (Y/F) expression vector (200 ng), the CREB dominant-negative mutant expression vector (200 ng) (vs. control vector) and pRcActin-LacZ (200 ng). At 48 h post-transfection, cells were harvested and assayed for luciferase activity. Luciferase activities were normalized over β-galactosidase activity as the mean ± S.E.M. from three independently transfected cells in the same experiment. Results are representative of three independent experiments. <b>(C)</b> BeWo cells were transfected with the CREB (Y/F) expression vector (vs. the control empty vector) (200 ng). At 48 h post-transfection, total RNA were extracted and analyzed by RT-PCR for both Syncytin-2 and β-actin mRNA. Band intensities of Syncytin-2 mRNA after normalization with β-actin mRNA levels are indicated below with values for pcDNA3.1-transfected cells set as 1. (<b>D-E</b>) BeWo cells were co-transfected with pGL3/600 (400 ng) or equivalent constructs bearing the M1 or M5 mutant (<b>D</b>) along with pCIneoCREB2 (or the empty vector pCIneo) and pRcActin-LacZ (200 ng). In (<b>E</b>), cells were either left untreated or treated with forskolin prior to lysis. Luciferase activities were normalized over β-galactosidase activity as the mean ± S.E.M. from three independently transfected cells in the same experiment. Results are representative of two independent experiments. (<b>F-G</b>) BeWo cells were treated or not with forskolin for 24 and 48 h and cell extracts were subsequently analyzed for CREB2 and GAPDH protein levels by Western blot. Band intensities for CREB2 are depicted in (<b>G</b>) following normalization with GAPDH signals and were calculated as the mean ratio ± S.E.M. from three independent experiments (*p< 0.05; **p< 0.01; ***p< 0.001).</p

Importance of the CRE/AP-1-like motif in induced Syncytin-2 promoter activity.

<p><b>(A-C)</b> BeWo cells were co-transfected with pGL3/600 (400 ng) or equivalent constructs bearing the M1 (<b>A</b>), M5 or M6 (<b>B</b>) mutant sequences along with pRcActin-LacZ (200 ng). In <b>C</b>, BeWo cells were co-transfected with pGL2-TATA-150 or pGL2-TATA-150M1 (vs. the control pGL2-TATA vector) (400 ng) along with pRcActin-LacZ (200 ng). Following transfection, cells were left untreated (Control or DMSO) or treated with forskolin (<b>A-C</b>) and RP-cAMP/SP-cAMP (<b>C</b>) for 12 h. (<b>D</b>) Primary villous cytotrophoblasts were microporated with pGL2-TATA-150 or pGL2-TATA-150M1 (vs. the control pGL2-TATA vector) (400 ng) along with pRcActin-LacZ (100 ng). Cells were then harvested and analyzed for luciferase expression. Luciferase values were normalized for β-galactosidase activity and are expressed as the mean normalized RLU ±S.E.M. from three independently transfected cells in the same experiment. Results in (<b>D</b>) are shown as mean of fold induction over pGL2-TATA-transfected cells (set as 1) and are representative of three independent experiments (**p< 0.01; ***p< 0.001). (<b>E</b>) Chromatin immunoprecipitation analysis of CREB1, CREB2 and JunD binding to Syncytin-2 promoter region in non-treated or treated BeWo cells was performed with antibodies specific to these factors or against Histone 3 (positive control) or with non-specific IgG antibodies (IgG: negative control). The-300/-150 region of the Syncytin-2 promoter was next PCR amplified on immunoprecipitated DNA. Input DNA was similarly amplified for control.</p

Identification of Syncytin-2 promoter regions responsive to forskolin induction in BeWo cells.

<p>The first 2600 bp region positioned upstream of the Syncytin-2 transcription initiation site in addition to the first 51 nucleotide of exon 1 were cloned upstream of the luciferase reporter gene. The resulting vector pGL3/2600 and successive 5’ deletion mutants vs. the control vector pGL3Basic (400 ng) were co-transfected with 200 ng pRcActin-LacZ in BeWo cells. Cells were either left untreated or treated with 50 μM forskolin. At 12 h post treatment, cells were harvested and assayed for luciferase activity in triplicates. Luciferase activities were normalized over β-galactosidase activity as the mean ± S.E.M. from three independently transfected cells in the same experiment. Results are shown as mean of fold induction of treated over untreated cell samples and are representative of four independent experiments (**p < 0.01 *** p < 0.001). The position of the previously identified GCM1-binding site and the transcription initiation site are both shown within the Syncytin-2 promoter in the pGL3/2600 construct.</p

Proposed model for CREB2/JunD-mediated activation of Syncytin-2 expression.

<p>Based on the experiments shown in this study, we propose that p38MAPK activation upon forskolin stimulation activates both CREB2 and JunD. These transcription factors bind to the CRE/AP-1 motif, possibly as an heterodimer. The specific binding of this complex to the Syncytin-2 promoter then recruits CBP/p300, which mediates histone acetylation and transcription. An unknown factor binds next to this region at a presumed GATA-binding site and might compete for binding, thereby acting negatively on CREB2/JunD-induced Syncytin-2 transcription. Possible alternatives for this model include the interaction of other CREB/ATF family members to the CRE/AP-1 motif.</p

Silencing of either JunD or CREB2 reduces Syncytin-2 expression and fusion of stimulated BeWo cells.

<p>BeWo cells were stimulated with forskolin or left untreated (DMSO) and transfected with 37,5 ng CREB-2- or JunD-specific siRNA vs. scrambled control siRNA. (<b>A, B</b>) Cell fusion index was calculated for each transfected cell samples by confocal microscopy analyzed with anti-desmoplakin antibodies and propidium iodide staining. (<b>C-D</b>) Western blot analyses were performed on cellular extracts from each transfected cell samples using anti-JunD, anti-CREB2 and anti-GAPDH antibodies (left panel). Analyses for Syncytin-2 proteins levels were normalized against GAPDH signals and band intensities are depicted in (<b>D</b>) (*p< 0.05; **p< 0.01).</p

Oligonucleotides used for construction of deleted mutants and site-directed mutagenesis of the Syncytin-2 promoter.

<p>Oligonucleotides used for construction of deleted mutants and site-directed mutagenesis of the Syncytin-2 promoter.</p