TSA-induced <i>TNFSF9</i> promoter activity depends on GC-boxes sequences.

<p>(A) The different pTNFSF9 (3)-luc mutated constructs were generated as described under “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007085#s2" target="_blank">Materials and Methods</a>”. The <i>hatched</i> boxes represent putative GC-boxes. The mutations in theses sites are symbolized by <i>filled</i> boxes. Jurkat cells were transiently cotransfected with the indicated reporter construct and the pRL-β to control for transfection efficiency. Cells were left unstimulated or treated with TSA (500 nM) for 16 hours. Means ± SD of three independent experiments are shown. Basal transcriptional activities were 7.7±1.8, 13.5±4.9, 96.7±19.2, 222.2±20.3, 133.5±7.6 and 175±81.7 RLU for the pGL3, pTNFSF9 (3) m4, pTNFSF9 (3) m3, pTNFSF9 (3) m2, pTNFSF9 (3) m1 and pTNFSF9 (3), respectively. (B) The indicated pTNFSF9 (3)-luc constructs were transfected in U937 (left panel) and Raji (right panel) cell lines and analyzed as described in (A). (C) The p-486TNFSF6/FasL promoter construct was transfected in Jurkat cells and analyzed as described in (A) except that PMA (20 ng/ml) and Ionomycin (1 µg/ml) were added, as indicated. Means ± SD of three independent experiments are shown. (D) RT-PCR was performed on total mRNA isolated from Jurkat cells pretreated with the GC-box binding inhibitor mithramycin A (MA) for 1 hour followed by incubation in the presence TSA (250 nM) for 8 hours. (E) Increasing concentrations of MA (100, 200 and 500 nM) was added to the cells 1 hour after transfection with the p<i>TNFSF9</i> (3) reporter plasmid, followed by the addition of TSA (500 nM) 1 hour later. The cells were harvested 16 hours after the addition of TSA and processed for luciferase assay. Means ± SD of three independent experiments are shown.</p

Induced proliferation of normal allogeneic leukocytes by Raji cells treated with HDACi.

<p>(A) FACS analysis of 4-1BBL expression on HDACi-treated Raji cells for 4, 8, 24 and 48 hours as indicated. Results are presented as % of positive cells. The data shown are from one experiment representative of a total of two. (B) PBMCs (2×10⁵) of healthy donor were incubated for 5 days with Raji cells (1×10⁵) treated or not for 24 hours with TSA (250 nM), MS-275 (2.5 µM) or SAHA (2.5 µM). PBMCs were stimulated or not with immobilized OKT3 (0,5 µg/ml) in the presence or absence of recombinant human 4-1BB-Fc (1 µg/ml) or control-Fc protein (1 µg/ml), as indicated, and proliferation was measured by incorporation of ³H-thymidine during the last 18 hours of culture. Supernatants from unstimulated (C) and from OKT3-stimulated (D) cell cultures were collected at 48 hours of culture and assayed for IFNγ by ELISA. Values are means of triplicates ± SD and this experiment is representative of two independent experiments. * p<0,05 and ** p<0,01 (t student). (E) As in (B), except that DR3-Fc recombinant protein was added (1 µM/ml), as indicated. Values are means of triplicates ± SD.</p

List of genes included in the quantitative RT-PCR low-density array.

<p>List of genes included in the quantitative RT-PCR low-density array.</p

Primers used for Electrophoretic Mobility Shift Assays.

<p>(Only forward primers are indicated).</p

Modulation of <i>TNFSF9</i> transcript levels by HDACi does not require <i>de novo</i> protein synthesis and does not require <i>TNFSF9</i> promoter region chromatin remodeling.

<p>Jurkat JA16 cells were left unstimulated (UN) or were incubated for 1 hour with CHX (10 µg/ml) and then treated for 8 hours by TSA (250 nM), MS-275 (1 µM) and SAHA (1 µM). Total mRNA was extracted and transcripts of <i>TNFSF9</i> and <i>GAPDH</i> were analysed by PCR (A) and quantified by real-time PCR (B) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007085#pone-0007085-g001" target="_blank">Fig. 1</a>. (C) Nuclei from Jurkat T cells left unstimulated (UN), or treated with TSA (500 nM) for 18 hours, were digested <i>in vivo</i> with increasing amounts of DNase I (0, 30, 40, 50, 60 and 70 U/ml) followed by <i>Sac</i>I digestion <i>in vitro</i> and indirect end-labeling (lower panel). The schematic organization of the <i>TNFSF9</i> locus, showing the indirect end-labeling and southern blotting strategy, as well as the DNaseI hypersensitive sites (DHS) identified in the course of the present study are depicted in the upper panel. The position of the probe used for the southern blotting, as well as the <i>TNFSF9</i> exon 1-3 are showed. Hypersensitive regions DHSI and DHSII are shown on the left. Molecular weight markers (MM) are a double digest of naked DNA by <i>Sac</i>I (12641 bp), <i>Nsi</i>I (9180 bp), <i>Nhe</i>I (6850 bp), <i>Hinc</i>II (5006 bp), <i>Xho</i>I (2390 bp) and <i>Bgl</i>II (1918 bp).</p

Sp proteins bind to the GC-box located in the <i>TNFSF9</i> promoter.

<p>(A) Nucleotide sequence of the three wild-type GC-box oligonucleotides probes are shown with underlined bases corresponding to the mutated bases. (B) The three wild-type GC-box oligonucleotides probes were incubated with nuclear extracts from Jurkat cells (10 µg) and treated or not for 4 hours with TSA (250 nM) in the absence or in the presence of unspecific competitor (dI-dC). The figure shows only the specific retarded bands of interest. (C) The three wild-type GC-box oligonucleotide probes were incubated with nuclear extracts from Jurkat cells (10 µg) in the absence (lane 2) or in the presence of the unlabeled oligonucleotide (lane 3) or of the mutated unlabeled oligonucleotide (lane 4). The figure shows only the specific retarded bands of interest. (D) Nuclear extracts from Jurkat cells treated or not for 4 hours with TSA (250 nM) were incubated in the absence of antibody or in the presence of antibodies directed against Sp1 and/or Sp3 (as indicated at the top of each lane) or with purified rabbit IgG as negative control, before addition of the oligonucleotide probe. The figure shows only the specific retarded bands of interest. Arrows indicate specific retarded DNA-protein complexes corresponding to Sp1 and Sp3. (E) SL2 cells were transiently cotransfected with pGL3 or p<i>TNFSF9</i> (3)-luc and with various amounts of the Sp1 expression construct (pHMW-Sp1) or the empty vector (pHMW) and processed for luciferase assays. Values were normalized relative to the protein concentration of the cellular lyzates. The experiment was repeated three times with similar results. <i>RLU</i>, relative light units.</p

Analysis of <i>TNFSF9</i> gene expression profiles induced by HDACi in leukaemia cell lines.

<p>(A) Jurkat JA16 cells were incubated with SAHA (lanes 2–5; 0.5, 1, 2.5 and 5 µM), MS-275 (lanes 6–9; 0.5, 1, 2.5 and 5 µM) or TSA (lanes 10–13, 50, 100, 200, 500 nM), or mock-treated (lane 1) for 16 hours, followed by cell lysis, SDS-PAGE and immunoblotting using the indicated antibodies (left panel). In parallel, Jurkat JA16 cells were incubated with SAHA (2 µM), TSA (200 nM), MS-275 (2 µM) or mock-treated (DMSO) for 6, 16 or 24 hours, as indicated, followed by cell lysis, SDS-PAGE and immunoblotting using the indicated antibodies (right panel). (B) Jurkat JA16 cells were incubated with TSA (500 nM), MS-275 (2,5 µM) or SAHA (2,5 µM) for 8 hours and left unstimulated or stimulated by PMA (20 ng/ml) and Ionomycin (1 µg/ml) for 4 hours. Following incubation time, total mRNA was extracted and analyzed by Transcription Low Density Array. Results obtained for <i>HPRT</i>, <i>βActin</i>, <i>p21/WAF/CDKN1A</i> and <i>TNFSF9</i> are presented as expression ratios relative to <i>GAPDH</i> transcript levels. The scale shows the level of expression, where red indicates increased gene expresssion, and the intensity of color correlated to the magnitude change. Black indicates no change. (C) mRNA from (B) were analyzed by RT-PCR, using <i>TNFSF9</i> and <i>GAPDH</i> primer pairs. (D) Jurkat JA16, RAJI and U937 cells were mock-treated or incubated with TSA (250 nM), MS-275 (1 µM) and SAHA (1 µM) for 4, 8 or 24 hours. Total mRNA was extracted and transcripts of <i>TNFSF9</i> and <i>GAPDH</i> were analyzed by RT-PCR. These conditions were compromized to compare for <i>TNFSF9</i> transcript detection and modulation by HDAC inhibition in different cell types producing distinct basal <i>TNFSF9</i> transcript levels.</p

Identification of TSA response elements in the human <i>TNFSF9</i> promoter.

<p>(A) DNA sequence of a 400-bp human <i>TNFSF9</i> promoter region. The ATG site is <i>boldface</i> (to which we assigned the position nucleotide + 1). Putative Sp1/Sp3 binding sites are boxed. Putative CCAAT boxes are underlined. (B) The constructs were generated by cloning progressively 5′-truncated human <i>TNFSF9</i> promoter fragments into the pGL3/basic luciferase vector. Negative numbers denote bp distances from translational start codon. (C) Jurkat cells were transiently cotransfected with the indicated reporter constructs and pRL-β to control for transfection efficiency. Transfected cells were either left untreated or treated with TSA (500 nM) for 16 hours. Data are the average ± SD of three independent experiments. <i>RLU</i>, relative light units.</p

New Pyridinone Derivatives as Potent HIV-1 Nonnucleoside Reverse Transcriptase Inhibitors

Several 5-ethyl-6-methyl-4-cycloalkyloxy-pyridin-2(1<i>H</i>)-ones were synthesized and evaluated for their anti HIV-1 activities against wild-type virus and clinically relevant mutant strains. A racemic mixture (<b>10</b>) with methyl substituents at positions 3 and 5 of the cyclohexyloxy moiety had potent antiviral activity against wild-type HIV-1. Subsequent stereoselective synthesis of a stereoisomer displaying both methyl groups in equatorial position was found to have the best EC₅₀. Further modulations focused on position 3 of the pyridinone ring improved the antiviral activity against mutant viral strains. Compounds bearing a 3-ethyl (<b>22</b>) or 3-isopropyl group (<b>23</b>) had the highest activity against wild-type HIV-1 and displayed low-nanomolar potency against several clinically relevant mutant strains

CTIP2 colocalizes with Sp1 and COUP-TF within Hp1α-associated structures

<p><b>Copyright information:</b></p><p>Taken from "COUP-TF interacting protein 2 represses the initial phase of HIV-1 gene transcription in human microglial cells"</p><p>Nucleic Acids Research 2005;33(7):2318-2331.</p><p>Published online 22 Apr 2005</p><p>PMCID:PMC1084325.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> () Microglial cells were transfected or not with Flag-CTIP2 as indicated. After being treated, endogenous Sp1, COUP-TF and Hp1α proteins were immunodetected with primary anti-COUP-TF (Santa Cruz Biotechnology) (images 1 and 2), anti-Sp1 (images 6 and 7) and anti-Hp1α antibodies (images 11 and 12). Overexpressed Flag-CTIP2 was detected with antibodies directed against the Flag epitope (images 3, 8 and 13). The primary immunocomplexes were revealed by CY2- or CY3-labeled anti-species secondary antibodies (green or red staining). Mask column (images 5, 10 and 15) shows the colocalized CY2 and CY3 stainings. () Microglial cells expressing RFP-CTIP2 (image 1) and GFP-Sp1 (image 3) were subjected to endogenous COUP-TF immunodetection with anti-COUP-TF antibodies (kindly provided by J. E. Mertz). COUP-TF immunocomplexes were stained by CY5- (blue staining) labeled anti-species secondary antibodies (image 2). Pattern of RFP-CTIP2 and GFP-Sp1 expressed alone are presented on images 5 and 6, respectively. ( and ) Coverslips were subjected to confocal microscopy analysis. Bar, 10 μm