Project description:Study the effect of PARP-14 and its activity on Th2 differentiation ChIP seq was performed on Th2 differentiated cells isolated from PARP-14 +/+ and PARP-14 -/- treated with or without PJ34
Project description:PARP-14, a member of the poly ADP-ribose polymerase super family, promotes T helper cell 2 (Th2) differentiation by regulating interleukin-4 (IL-4) and STAT6-dependent transcription. Yet, whether PARP-14 globally impacts gene regulation has not been determined. In this report, using an RNA pol II ChIP-seq approach, we identify genes in Th2 cells that are regulated by PARP-14, and either dependent or independent of ADP-ribosyltransferase catalytic activity. Our data demonstrate that PARP-14 enhances the expression of Th2 genes as it represses the expression of Th1-associated genes. Among the relevant targets are Signal Transducer and Activator of Transcription genes required for polarizing Th1 and Th2 cells. To define a mechanism for PARP-14 function, we use an informatics approach to identify putative PARP-14 DNA binding sites. Two putative PARP-14 binding motifs are identified in multiple Th2 cytokine genes, and we demonstrate that PARP-14 interacts with each motif using in vitro binding assays. Taken together our results indicate that PARP-14 is an important factor for T helper cell differentiation and it binds to specific DNA sequences to mediate its function.
Project description:Poly(ADP-ribose) polymerase-2 (PARP-2) is acknowledged as a DNA repair enzyme; however, recently metabolic properties had been attributed to it. Hereby, we examined the metabolic consequences of PARP-2 ablation in liver. Microarray analysis of PARP-2 knockdown HepG2 cells revealed the dysregulation of lipid and cholesterol metabolism genes. Induction of cholesterol biosynthesis genes stemmed from the enhanced expression of sterol-regulatory element binding protein (SREBP)-1. We revealed that PARP-2 is a suppressor of the SREBP-1 promoter, therefore ablation of PARP-2 induces SREBP-1 expression and consequently cholesterol synthesis. PARP-2-/- mice had higher SREBP-1 expression that was translated into enhanced hepatic and serum cholesterol levels. PARP-2 silencing was performed employing shPARP-2 (small hairpin) and scPARP-2 (scrambled) shRNA by lentiviral delivery (Sigma) using 40 MOI lentiviruses coding shRNA sequence against PARP-2.
Project description:Adenylate cyclase signaling pathway is suggested to be a key regulator of immune system functions. However, specific effects of cyclic adenosine monophosphate on T helper cell differentiation and functions are unclear. Involvement of cAMP in Th cell differentiation program, in particular development of Th1, Th2, and Th17 subsets, was evaluated employing forskolin (FSK), a labdane diterpene well known as AC activator.
Project description:We are examining the consequences of activating mutations in PI3K-delta in Th1 vs. Th2 differentiation. Using the house dust mite (HDM) model of allergic airway inflammation, which drives potent Th2 responses in vivo, we have found mice expressing hyper-activated PI3K-delta show impaired Th2 differentiation and instead favor Th1 cell differentiation. We would like to further investigate this aberrant CD4 differentiation profile using scRNA-seq to characterize PI3K-delta regulated aspects of type II immunity.
Project description:Eukaryotic gene regulation implies that transcription factors gain access to genomic information via poorly understood processes involving activation and targeting of kinases, histone-modifying enzymes, and chromatin remodelers to chromatin. Here we report that progestin gene regulation in breast cancer cells requires a rapid and transient increase in poly-(ADP)-ribose (PAR), accompanied by a dramatic decrease of cellular NAD that could have broad implications in cell physiology. This rapid increase in nuclear PARylation is mediated by activation of PAR polymerase PARP-1 as a result of phosphorylation by cyclin-dependent kinase CDK2. Hormone-dependent phosphorylation of PARP-1 by CDK2, within the catalytic domain, enhances its enzymatic capabilities. Activated PARP-1 contributes to the displacement of histone H1 and is essential for regulation of the majority of hormone-responsive genes and for the effect of progestins on cell cycle progression. Both global chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) and gene expression analysis show a strong overlap between PARP-1 and CDK2. Thus, progestin gene regulation involves a novel signaling pathway that connects CDK2-dependent activation of PARP-1 with histone H1 displacement. Given the multiplicity of PARP targets, this new pathway could be used for the pharmacological management of breast cancer. PARP-1 activation mechanism by CDK2 in response of progestin in breast cancer cells
Project description:Allergic asthma is a T helper 2 (Th2) cell-associated inflammatory disease, driven by cytokines such as IL-4, IL-5, and IL-13. Th2 cells express the G-protein-coupled receptor CRTh2, a receptor for prostaglandin D2 (PGD2) that influences Th2 function and survival. Inhaled glucocorticosteroids are the primary treatment of allergic asthma and improve asthma symptoms by inhibiting Th2 cytokine production. Women are more likely than men to have severe asthma and to have symptoms requiring a hospital visit. These findings lead us to consider a mechanism by which female sex hormones could influence Th2 cell response to glucocorticosteroid. Using whole-mRNA sequencing, we examined gene expression in primary Th2 cells following exposure to glucocorticosteroids (0.1µM) in the presence or absence of an estrogen mimic, PPT (10µM).
Project description:ChIP-seq was performed to analyze genome-wide distribution of PARP-1 and histone marks H3K4me3, H3K27me3 in mES cells, and examine the effect of PARP-1 knockout on Sox2 and Oct4 genome-wide distribution in ES cells.