Activation of LTRs from Different Human Endogenous Retrovirus (HERV) Families by the HTLV-1 Tax Protein and T-Cell Activators

Human endogenous retroviruses (HERVs) represent approximately 8% of our genome. HERVs influence cellular gene expression and contribute to normal physiological processes such as cellular differentiation and morphogenesis. HERVs have also been associated with certain pathological conditions, including cancer and neurodegenerative diseases. As HTLV-1 causes adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and has been shown to modulate host gene expression mainly through the expression of the powerful Tax transactivator, herein we were interested in looking at the potential modulation capacity of HTLV-1 Tax on HERV expression. In order to evaluate the promoter activity of different HERV LTRs, pHERV-LTR-luc constructs were co-transfected in Jurkat T-cells with a Tax expression vector. Tax expression potently increased the LTR activity of HERV-W8 and HERV-H (MC16). In parallel, Jurkat cells were also stimulated with different T-cell-activating agents and HERV LTRs were observed to respond to different combination of Forskolin, bpV[pic] a protein tyrosine phosphatase inhibitor, and PMA. Transfection of expression vectors for different Tax mutants in Jurkat cells showed that several transcription factors including CREB appeared to be important for HERV-W8 LTR activation. Deletion mutants were derived from the HERV-W8 LTR and the region from −137 to −123 was found to be important for LTR response following Tax expression in Jurkat cells, while a different region was shown to be required in cells treated with activators. Our results thus demonstrated that HTLV-1 Tax activates several HERV LTRs. This raises the possibility that upregulated HERV expression could be involved in diseases associated with HTLV-1 infection

Activation of LTRs from Different Human Endogenous Retrovirus (HERV) Families by the HTLV-1 Tax Protein and T-Cell Activators

Human endogenous retroviruses (HERVs) represent approximately 8% of our genome. HERVs influence cellular gene expression and contribute to normal physiological processes such as cellular differentiation and morphogenesis. HERVs have also been associated with certain pathological conditions, including cancer and neurodegenerative diseases. As HTLV-1 causes adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and has been shown to modulate host gene expression mainly through the expression of the powerful Tax transactivator, herein we were interested in looking at the potential modulation capacity of HTLV-1 Tax on HERV expression. In order to evaluate the promoter activity of different HERV LTRs, pHERV-LTR-luc constructs were co-transfected in Jurkat T-cells with a Tax expression vector. Tax expression potently increased the LTR activity of HERV-W8 and HERV-H (MC16). In parallel, Jurkat cells were also stimulated with different T-cell-activating agents and HERV LTRs were observed to respond to different combination of Forskolin, bpV[pic] a protein tyrosine phosphatase inhibitor, and PMA. Transfection of expression vectors for different Tax mutants in Jurkat cells showed that several transcription factors including CREB appeared to be important for HERV-W8 LTR activation. Deletion mutants were derived from the HERV-W8 LTR and the region from −137 to −123 was found to be important for LTR response following Tax expression in Jurkat cells, while a different region was shown to be required in cells treated with activators. Our results thus demonstrated that HTLV-1 Tax activates several HERV LTRs. This raises the possibility that upregulated HERV expression could be involved in diseases associated with HTLV-1 infection

A CRE/AP-1-like motif is essential for induced syncytin-2 expression and fusion in human trophoblast-like model.

Syncytin-2 is encoded by the envelope gene of Endogenous Retrovirus-FRD (ERVFRD-1) and plays a critical role in fusion of placental trophoblasts leading to the formation of the multinucleated syncytiotrophoblast. Its expression is consequently regulated in a strict manner. In the present study, we have identified a forskolin-responsive region located between positions -300 to -150 in the Syncytin-2 promoter region. This 150 bp region in the context of a minimal promoter mediated an 80-fold induction of promoter activity following forskolin stimulation. EMSA analyses with competition experiments with nuclear extracts from forskolin-stimulated BeWo cells demonstrated that the -211 to -177 region specifically bound two forskolin-induced complexes, one of them containing a CRE/AP-1-like motif. Site-directed mutagenesis of the CRE/AP-1 binding site in the context of the Syncytin-2 promoter or a heterologous promoter showed that this motif was mostly essential for forskolin-induced promoter activity. Transfection experiments with dominant negative mutants and constitutively activated CREB expression vectors in addition to Chromatin Immunoprecipitation suggested that a CREB family member, CREB2 was binding and acting through the CRE/AP-1 motif. We further demonstrated the binding of JunD to this same motif. Similar to forskolin and soluble cAMP, CREB2 and JunD overexpression induced Syncytin-2 promoter activity in a CRE/AP-1-dependent manner and Syncytin-2 expression. In addition, BeWo cell fusion was induced by both CREB2 and JunD overexpression, while being repressed following silencing of either gene. These results thereby demonstrate that induced expression of Syncytin-2 is highly dependent on the interaction of bZIP-containing transcription factors to a CRE/AP-1 motif and that this element is important for the regulation of Syncytin-2 expression, which results in the formation of the peripheral syncytiotrophoblast layer

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

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

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

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

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

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