Poly(ADP-ribose) polymerase 1 is necessary for coactivating hypoxia-inducible factor-1-dependent gene expression by Epstein-Barr virus latent membrane protein 1

Abstract

Latent membrane protein 1 (LMP1) is the major transforming protein of Epstein-Barr virus (EBV) and is critical for EBV-induced B-cell transformation in vitro. Poly(ADP-ribose) polymerase 1 (PARP1) regulates accessibility of chromatin, alters functions of transcriptional activators and repressors, and has been directly implicated in transcriptional activation. Previously we showed that LMP1 activates PARP1 and increases Poly(ADP-ribos)ylation (PARylation) through PARP1. Therefore, to identify targets of LMP1 that are regulated through PARP1, LMP1 was ectopically expressed in an EBV-negative Burkitt’s lymphoma cell line. These LMP1-expressing cells were then treated with the PARP inhibitor olaparib and prepared for RNA sequencing. The LMP1/PARP targets identified through this RNA-seq experiment are largely involved in metabolism and signaling. Interestingly, Ingenuity Pathway Analysis of RNA-seq data suggests that hypoxia-inducible factor 1-alpha (HIF-1α) is an LMP1 target mediated through PARP1. PARP1 is acting as a coactivator of HIF-1α-dependent gene expression in B cells, and this co-activation is enhanced by LMP1-mediated activation of PARP1. HIF-1α forms a PARylated complex with PARP1 and both HIF-1α and PARP1 are present at promoter regions of HIF-1α downstream targets, leading to accumulation of positive histone marks at these regions. Complex formation, PARylation and binding of PARP1 and HIF-1α at promoter regions of HIF-1α downstream targets can all be attenuated by PARP1 inhibition, subsequently leading to a buildup of repressive histone marks and loss of positive histone marks. In addition, LMP1 switches cells to a glycolytic ‘Warburg’ metabolism, preferentially using aerobic glycolysis over mitochondrial respiration. Finally, LMP1+ cells are more sensitive to PARP1 inhibition and, therefore, targeting PARP1 activity may be an effective treatment for LMP1+ EBV-associated malignancies.

Epstein–Barr virus-encoded latent membrane protein 1 and B-cell growth transformation induces lipogenesis through fatty acid synthase

“…1C). This may partly explain the ability of olaparib to blunt the proliferative advantage bestowed by LMP1 that we previously reported (9). Finally, in a comparison of LMP1+ DG75s treated with olaparib compared to untreated pBABE, we found that each metabolite's fold changes are roughly 50% less than those observed in LMP1+ untreated cells compared to pBABE.…”

“…To identify cellular metabolites that can be altered by LMP1, we first ectopically expressed LMP1 in the EBV-negative Burkitt's lymphoma (BL) cell line DG75. Cells were transduced with retro-viral particles containing either pBABE-HA (empty vector) or pBABE-HA-LMP1 (LMP1) vectors as described previously (9). Using this cell system, we then undertook a targeted approach to determine the relative quantities of approximately 200 polar metabolites spanning 32 different classes to examine LMP1induced metabolic changes.…”

“…1C). Previously, we have shown Poly(ADP-Ribose) Polymerase 1 to be important in LMP1-induced aerobic glycolysis and accelerated cellular proliferation using the PARP inhibitor olaparib (9). Therefore, we included an olaparib treatment group in our metabolic analysis to examine whether PARP inhibition could offset LMP1-induced changes to cellular metabolites.…”

“…The activation of these signaling pathways by LMP1 contribute to its ability to transform cells by altering the expression of a wide range of host gene targets (7). LMP1 has also been shown to promote aerobic glycolysis and metabolic reprogramming in B cell lymphomas and nasopharyngeal epithelial cells (8)(9)(10)(11)(12)(13)(14). The transition from a resting B-cell to a rapidly proliferating cell following EBV infection, and the presence of EBV in associated malignancies, entails major metabolic changes.…”

Dichloroacetate restores colorectal cancer chemosensitivity through the p53/miR-149-3p/PDK2-mediated glucose metabolic pathway

“…Next day, cells medium was changed to bicarbonate-free low-buffered assay medium supplemented with glucose, sodium pyruvate, and glutamin. After cells incubated for 1 h at 37°C in a non-CO 2 incubator, oxygen consumption rate and extracellular acidification rate were measured before and after the injection of DCA/ctrl, Rotenone (Rot) + antimycin A (AA) and 2deoxy-d-glucose (2-DG) using the Seahorse XF instrument (Agilent, Santa Clara, CA) as previously described [59,60]. Experiments were performed in real time in five to six replicate wells.…”