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Background/objectives: Euchromatic histone lysine methyltransferase 2 (EHMT2, also known as G9a) is a mammalian histone methyltransferase that catalyzes the dimethylation of histone 3 lysine 9 (H3K9). On human chromosome 15, the parental-specific expression of Prader–Willi Syndrome (PWS)-related genes, such as SNRPN and SNORD116, are regulated through the genetic imprinting of the PWS imprinting center (PWS-IC). On the paternal allele, PWS genes are expressed whereas the epigenetic maternal silencing of PWS genes is controlled by the EHMT2-mediated methylation of H3K9 in PWS-IC. Here, we measured the effects of RNA interference of EHMT2 on the maternal expression of genes deficient in PWS in mouse model and patient iPSC-derived cells. Methods: We used small interfering RNA (siRNA) oligonucleotides and lentiviral short harpin RNA (shRNA) to reduce Ehtm2/EHMT2 expression in mouse Snord116 deletion primary neurons, PWS patient-derived induced pluripotent stem cell (iPSC) line and PWS iPSC-derived neurons. We then measured the expression of transcript or protein (if relevant) of PWS genes normally silenced on the maternal allele. Results: With an approximate reduction of 90% in EHMT2 mRNA and more than 80% of the EHMT2 protein, we demonstrated close to a 2-fold increase in the expression of maternal transcripts for SNRPN and SNORD116 in PWS iPSCs treated with siEHMT2 compared to PWS iPSC siControl. A similar increase in SNORD116 and SNRPN RNA expression was observed in PWS iPSC-derived neurons treated with shEHMT2. Conclusions: RNAi reduction in EHMT2 activates maternally silenced PWS genes. Further studies are needed to determine whether the increase is therapeutically relevant. This study confirms the role of EHMT2 in the epigenetic regulation of PWS genes. [ABSTRACT FROM AUTHOR]

Unlabelled: In Kaposi's sarcoma-associated herpesvirus (KSHV), poly(ADP-ribose) polymerase 1 (PARP-1) acts as an inhibitor of lytic replication. Here, we demonstrate that KSHV downregulated PARP-1 upon reactivation. The viral processivity factor of KSHV (PF-8) interacted with PARP-1 and was sufficient to degrade PARP-1 in a proteasome-dependent manner; this effect was conserved in murine gammaherpesvirus 68. PF-8 knockdown in KSHV-infected cells resulted in reduced lytic replication upon reactivation with increased levels of PARP-1, compared to those in control cells. PF-8 overexpression reduced the levels of the poly(ADP-ribosyl)ated (PARylated) replication and transcription activator (RTA) and further enhanced RTA-mediated transactivation. These results suggest a novel viral mechanism for overcoming the inhibitory effect of a host factor, PARP-1, thereby promoting the lytic replication of gammaherpesvirus., Importance: Gammaherpesviruses are important human pathogens, as they are associated with various kinds of tumors and establish latency mainly in host B lymphocytes. Replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is a central molecular switch for lytic replication, and its expression is tightly regulated by many host and viral factors. In this study, we investigated a viral strategy to overcome the inhibitory effect of poly(ADP-ribose) polymerase 1 (PARP-1) on RTA's activity. PARP-1, an abundant multifunctional nuclear protein, was downregulated during KSHV reactivation. The viral processivity factor of KSHV (PF-8) directly interacted with PARP-1 and was sufficient and necessary to degrade PARP-1 protein in a proteasome-dependent manner. PF-8 reduced the levels of PARylated RTA and further promoted RTA-mediated transactivation. As this was also conserved in another gammaherpesvirus, murine gammaherpesvirus 68, our results suggest a conserved viral modulation of a host inhibitory factor to facilitate its lytic replication., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The inflammasome is a molecular platform that stimulates the activation of caspase-1 and the processing of pro-interleukin (IL)-1β and pro-IL-18 for secretion. The NOD-like receptor family, pyrin domain containing 3 (NLRP3) protein is activated by diverse molecules and pathogens, leading to the formation of the NLRP3 inflammasome. Recent studies showed that the NLRP3 inflammasome mediates innate immunity against influenza A virus (IAV) infection. In this study, we investigated the function of the IAV non-structural protein 1 (NS1) in the modulation of NLRP3 inflammasome. We found that NS1 proteins derived from both highly pathogenic and low pathogenic strains efficiently decreased secretion of IL-1β and IL-18 from THP-1 cells treated with LPS and ATP. NS1 overexpression significantly impaired the transcription of proinflammatory cytokines by inhibiting transactivation of the nuclear factor-κB (NF-κB), a major transcription activator. Furthermore, NS1 physically interacted with endogenous NLRP3 and activation of the NLRP3 inflammasome was abrogated in NS1-expressing THP-1 cells. These findings suggest that NS1 downregulates NLRP3 inflammasome activation by targeting NLRP3 as well as NF-κB, leading to a reduction in the levels of inflammatory cytokines as a viral immune evasion strategy.