Project description:Many cytokines are involved in the pathogenesis of autoimmune diseases and are recognized as relevant therapeutic targets to attenuate inflammation, such as TNFα in RA and IFNα/γ in SLE. To relate the transcriptional imprinting of cytokines in a cell type-specific and disease-specific manner, we generated gene-expression profiles from peripheral monocytes of SLE and RA patients and compared them to in vitro-generated signatures induced by TNFα, IFNα2a and IFNγ. Monocytes from SLE and RA patients revealed disease-specific gene-expression profiles. In vitro-generated signatures induced by IFNα2a and IFNγ showed similar profiles that only partially overlapped with those induced by TNFα. Comparisons between disease-specific and in vitro-generated signatures identified cytokine-regulated genes in SLE and RA with qualitative and quantitative differences. The IFN-responses in SLE and RA were found to be regulated in a STAT1-dependent and STAT1-independent manner, respectively. Similarly, genes recognized as TNFα-regulated were clearly distinguishable between RA and SLE patients. While the activity of SLE monocytes was mainly driven by IFN, the activity from RA monocytes showed a dominance of TNFα that was characterized by STAT1 down-regulation. The responses to specific cytokines were revealed to be disease-dependent and reflected the interplay of cytokines within various inflammatory milieus. This study has demonstrated that monocytes from RA and SLE patients exhibit disease-specific gene-expression profiles, which can be molecularly dissected when compared to in vitro-generated cytokine signatures. The results suggest that an assessment of cytokine-response status in monocytes may be helpful for improvement of diagnosis and selection of the best cytokine target for therapeutic intervention. Expression profiles of human peripheral blood monocytes activated in vivo and stimulated in vitro. Monocytes from patients with SLE and RA and from healthy donors were used for generating disease-specific gene-expression profiles, where these profiles represent in vivo activation of monocytes. In addition, monocytes from healthy donors were stimulated in vitro by cytokines: TNFα, IFNα2a and IFNγ. Cytokine-specific gene-expression profiles were generated by comparing stimulated monocytes with unstimulated ones. TNFα-, IFNα2a- and IFNγ as cytokine-specific gene-expression profiles were compared with RA and SLE, as disease-specific gene-expression profiles.

Project description:Poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG) are enzymes that modify target proteins in the nucleus by the addition and removal, respectively, of ADP-ribose polymers. Although a role for PARP-1 in gene regulation has been well established, the role of PARG is less clear. To investigate how PARP-1 and PARG coordinately regulate global patterns of gene expression, we used short hairpin RNAs (shRNAs) to stably knockdown PARP-1 or PARG in MCF-7 cells, followed by expression microarray analyses. Experiment Overall Design: Two shRNAs targeting Luciferase (Luc, control), PARP-1, or PARG were cloned into the pSUPER.retro vector (puro or neo resistant) and sequentially introduced into parental MCF-7 cells using a standard retroviral infection technique. Luc, PARP-1, and PARG control-matched cell lines were generated three independent times. Experiment Overall Design: Total RNA was isolated from stable Luc, PARP-1, and PARG knockdown cells was analyzed for global patterns of gene expression. Briefly, 7 Î¼g of total RNA was labeled using Affymetrix's standard one-cycle amplification and labeling protocol. The labeled cRNA was then hybridized to Affymetrix Human U133A 2.0 GeneChips, which were scanned using a GeneChip Scanner 3000. Each biological replicate (1, 2 or 3) represents a single RNA isolation from each set of cell lines. Experiment Overall Design: These 9 samples were samples were part of a 15-sample microarray analysis (GSE12971) examining expression regulation by SIRT1, PARP-1, PARG and macroH2A. All 15 samples were included for the data normalization steps. Experiment Overall Design: The raw array data was processed by Affymetrix GeneChip Operating Software (GCOS) to obtain detection calls (ABS_CALL) and signal values (VALUE_2). The signals were then normalized by scaling to a target value of 500 using GCOS. To adjust for batch effects due to day-to-day differences in RNA isolations, the empirical Bayes method was applied to the data set. After adjusting any values less than 0.01 to 0.01, the data was log2 transformed, median centered for each array, and median centered for each individual probe set (VALUE). Filters were then applied to obtain final gene lists. Experiment Overall Design: Specifically, the criteria for a gene to be considered regulated by PARP-1 or PARG was: (1) detection call flagged as present or marginal in 2 out of 3 array replicates for both control and factor knockdown cell lines and (2) significance of values between control and knockdown cell lines for any given gene had a two-tailed Studentâ??s t-test with a p-value < 0.05. To determine the genes most robustly regulated by PARP-1 or PARG, a fold change criteria was applied, namely log2 fold change > 0.5 or < -0.5 when compared to the Luc control knockdown cells.

Project description:Vitamin A (retinol) is an essential precursor for the production of retinoic acid (RA), which in turn is a major regulator of gene expression, affecting cell differentiation throughout the body. Understanding how vitamin A nutritional status, as well as therapeutic retinoid treatment, regulates the expression of retinoid homeostatic genes is important for improving dietary recommendations and therapeutic strategies using retinoids. This study investigated genes central to processes of retinoid uptake and storage, release to plasma, and oxidation in the liver of rats under steady-state conditions after different exposures to dietary vitamin A (deficient, marginal, adequate and supplemented), and acutely after administration of a therapeutic dose of all-trans-RA. Over a very wide range of dietary vitamin A, lecithin:retinol acyltransferase (LRAT) as well as multiple cytochrome P450s (CYP26A1, CYP26B1, and CYP2C22) differed by diet and were highly correlated with one another and with vitamin A status assessed by liver retinol concentration (all correlations, P<0.05). After acute treatment with RA, the same genes were rapidly and concomitantly induced, preceding RARß, a classical direct target of RA. CYP26A1 mRNA exhibited the greatest dynamic range (change of log26 in 3 h). Moreover, CYP26A1 increased more rapidly in the liver of RA-primed rats than naïve rats. By in situ hybridization, CYP26A1 mRNA was strongly regulated within hepatocytes, closely resembling RBP4 in location. Overall, whether RA is produced endogenously from retinol or administered exogenously, changes in retinoid homeostatic gene expression simultaneously favor both retinol esterification and RA oxidation, with CYP26A1 exhibiting the greatest dynamic change. All rats were housed in a room maintained at 22°C with a 12-h dark:light cycle, and food and water were freely available. For the Steady-State Vitamin A Study (experiment 1) and the Retinoic Acid 16-hour Kinetic Study (experiment 2), lactating female Sprague-Dawley rats with 12 female pups (purchased from Charles River Laboratories, Willmington, MA) were fed a vitamin A-deficient purified diet [AIN-93G diet, prepared by Research Diets, New Brunswick, NJ] to reduce the transfer of vitamin A in milk from mother to pups prior to the start of the study. For experiment 1, from weaning, the offspring were fed the same diet modified to contain vitamin A at one of four levels: 0 (vitamin A deficient), 0.4 mg retinol/kg diet (vitamin A marginal), 4 mg retinol/kg diet (vitamin A adequate control), or 100 mg retinol/kg diet (vitamin A supplemented). All rats were studied at 8 weeks of age. Rats were euthanized by carbon dioxide asphyxiation and blood and liver were collected rapidly and frozen in liquid nitrogen for storage at -80°C before analysis. In experiment 2, at 8 weeks of age female vitamin A-deficient rats were treated with ~100 ug of All-trans-Retinoic acid (at-RA) for 0 (vehicle only), 3, 6, 10 or 16 h (n=3-4/group). Tissues were collected and RNA was prepared in the same manner as in experiment 1. In the 90-minute "first pass" kinetic study (experiment 3), female rats were purchased at 6 weeks of age and fed a stock rodent diet. When the rats were 8 weeks old they were assigned to a control (naïve) group. Rats in the naive group received an equal amount of vehicle only (vegetable oil/5% ethanol). Food was removed immediately. Sixteen hours after priming, each rat was lightly anesthetized by isoflurane-oxygen inhalation and treated with ~25 ug all-trans-RA bound to albumin [10 ug RA per 100 g bo dy weight], injected into the exposed left common iliac vein. The incision was closed with a surgical staple. The rats were allowed to recover from the anesthesia. Rats were killed at 0 minute (vehicle injection), and 30, 60, and 90 min (n = 3/group) after injection of the RA test dose. Rats were euthanized by carbon dioxide asphyxiation and blood and liver were collected rapidly and frozen in liquid nitrogen for storage at -80°C before analysis. For experiment 1, five biological repeats were Vitamin A deficient, seven biological repeats were Vitamin A marginal, six biological repeats were Vitamin A adequate, and two biological repeats were Vitamin A supplemented. In experiment 2, three biological repeats were performed for each of the following treatments: untreated, at-RA for 3 hours, at-RA for 6 hours and at-RA for 16 hours. Four biological repeats were treated with at-RA for 10 hours. For experiment 3, two biological repeats were untreated and three biological repeats were performed for each of the following at-RA treatment times: 30, 60 and 90 minutes. A total of 47 samples were analyzed.

Project description:Cell-and context-specific activities of nuclear receptors may in part be due to distinct coregulator complexes recruited to distinct subsets of target genes. RIP140 (also called NRIP1) is a ligand-dependent corepressor that is inducible with retinoic acid (RA). We have shown previously that silencing of RIP140 enhances RA-induced differentiation and enhances the induction of model RA target genes in human embryonal carcinoma cells (EC). Through use of microarray technology we sought to elucidate in a de novo fashion the global role of RIP140 in RA-dependent signaling. RIP140-dependent gene expression was largely consistent with RIP140 functioning to limit RAR signaling. Few if any genes were regulated in a manner to support a role for RIP140 in â??active repressionâ??. Interestingly, approximately half of the RA-dependent genes were unaffected by RIP140, suggesting that RIP140 may discriminate between different classes of RA target genes. RIP140 silencing also accelerated RA target gene activation and sensitized EC cells to low doses of RA. Together the data suggests that the RIP140-dependent RA target genes identified here may be particularly important in mediating RA-induced tumor cell differentiation. RIP140 may be an attractive target to sensitize tumor cells to retinoid-based differentiation therapy. Experiment Overall Design: The current investigation sought to determine the global effects of RIP140 on RA-dependent and RAâ??independent gene expression. A dose of siRNA of 90 nM was chosen. Twelve independent hybridizations were performed from mRNA isolated from 12 independent samples. One scrambled and two independent RISC-free siRNA transfections and transfections with three distinct RIP140 siRNAs were performed in NT2/D1 cells. Each siRNA treatment was further treated with either 10 Î¼M RA or DMSO control for 24 hours. Hence, the design consisted of four groups, the three siRNA controls treated with vehicle, Group 1; the siRNA controls treated with RA, Group 2; siRNA to RIP140 treated with vehicle, Group 3; and siRNA to RIP140 treated with RA, Group 4 . Experiment Overall Design: The treatments were done in the presence of normal sera, since charcoal absorption of sera removes many small hydrophobic ligands to the nuclear receptor family in addition to retinoids. This measure was taken with the aim to perhaps identify target genes of other nuclear receptors including orphan receptors that could be regulated by RIP140 in the presence of low levels of their cognate ligands. RA treatment of 10 Î¼M for 24 hours was chosen since previous experience with NT2/D1 cells indicated that a reasonably sized set of altered genes would be obtained. In addition, NT2/D1 cells commit to RA-induced differentiation by 48 hours and RIP140 repression enhances and accelerates the effects of RA in these cells. Therefore, the 24-hour time point is well-within the range of the RA commitment â??windowâ?? and a larger percentage of direct RA-target genes could be expect to be uncovered compared to later time points. Experiment Overall Design: Based on the current understanding of the properties of RIP140, a number of potential outcomes were anticipated: 1) RIP140 may limit RA target gene activation by negative-feedback inhibition upon RA addition, 2) RIP140 may participate in â??active repressionâ?? of gene expression upon RA addition, 3) an unexpected outcome that RIP140 may participate in the activation of a subset of target genes, 4) RIP140 may regulate the transcription of target genes of other nuclear receptors or even non-nuclear receptor transcription factors. Experiment Overall Design: Total RNA was purified using RNeasy columns (Qiagen). Hybridizations were performed according to Affymetrix guidelines at the Dartmouth College Microarray Shared Resource using an Affymetrix GeneChip Workstation. Biotin-labeled cRNA was generated from 5 Î¼g of total RNA and hybridized to the Human Genome (HG) U133 Plus 2.0 chip A total of 12 hybridizations were performed comprising 12-independent biologic samples organized into the four groups of three.

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Gene expression before or 3 hours after t-RA treatment in HeLa cells expressing an shRNA control or shRNA against PARG

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