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Polymerase independent repression of FoxO1 transcription by sequence-specific PARP1 binding to FoxO1 promoter.

ABSTRACT: Poly(ADP-ribose) polymerase 1 (PARP1) regulates gene transcription in addition to functioning as a DNA repair factor. Forkhead box O1 (FoxO1) is a transcription factor involved in extensive biological processes. Here, we report that PARP1 binds to two separate motifs on the FoxO1 promoter and represses its transcription in a polymerase-independent manner. Using PARP1-knock out (KO) cells, wild-type-PARP1-complemented cells and catalytic mutant PARP1^E988K-reconstituted cells, we investigated transcriptional regulation by PARP1. PARP1 loss led to reduced DNA damage response and ~362-fold resistance to five PARP inhibitors (PARPis) in Ewing sarcoma cells. RNA sequencing showed 492 differentially expressed genes in a PARP1-KO subline, in which the FoxO1 mRNA levels increased up to more than five times. The change in the FoxO1 expression was confirmed at both mRNA and protein levels in different PARP1-KO and complemented cells. Moreover, exogenous PARP1 overexpression reduced the endogenous FoxO1 protein in RD-ES cells. Competitive EMSA and ChIP assays revealed that PARP1 specifically bound to the FoxO1 promoter. DNase I footprinting, mutation analyses, and DNA pulldown FREP assays showed that PARP1 bound to two particular nucleotide sequences separately located at -813 to -826 bp and -1805 to -1828 bp regions on the FoxO1 promoter. Either the PARPi olaparib or the PARP1 catalytic mutation (E988K) did not impair the repression of PARP1 on the FoxO1 expression. Exogenous FoxO1 overexpression did not impair cellular PARPi sensitivity. These findings demonstrate a new PARP1-gene promoter binding mode and a new transcriptional FoxO1 gene repressor.

SUBMITTER: Tian YN

PROVIDER: S-EPMC6987093 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Influenza viruses encode a viral RNA-dependent RNA polymerase (FluPol), which is responsible for transcribing and replicating the negative-sense viral RNA (vRNA) genome. FluPol transcribes vRNA using a host-capped mRNA primer and replicates it by synthesizing a positive-sense cRNA intermediate, which is copied back into vRNA. To carry out these functions, FluPol interacts with vRNA and cRNA using conserved promoter elements at the 5' and 3' termini. Recent structural studies have identified a new surface binding site for the 3' vRNA and cRNA promoters on FluPol, referred to as the mode B site. However, the role of this binding site in FluPol function is unknown. In this study, we used a combination of cell-based and biochemical assays to show that the mode B site is important for both viral genome transcription and replication in influenza A virus. Furthermore, we show that the mode B site is not needed for initiating transcription in vitro but is required to synthesize a full-length product. This is consistent with a model in which the 3' terminus of the vRNA template binds in the mode B site during elongation. Our data provide the first functional insights into the role of the mode B site on FluPol, which advances our understanding of FluPol function and influenza virus replication.IMPORTANCE Influenza viruses are responsible for up to 650,000 deaths per year through seasonal epidemics, and pandemics have caused tens of millions of deaths in the past. Most current therapeutics suffer from widespread resistance, creating a need for new drug targets against influenza virus. The virus encodes an RNA-dependent RNA polymerase, which replicates and transcribes the vRNA genome. The polymerase interacts with vRNA and the complementary replicative intermediate cRNA using several specific binding sites; however, the functions associated with these binding sites remain unknown. Here, we functionally characterize a binding site for the 3' vRNA and cRNA promoters. Our data offer insight into the mechanism of viral genome transcription by the influenza virus polymerase and may be applicable to other related viruses.

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Polymerase independent repression of FoxO1 transcription by sequence-specific PARP1 binding to FoxO1 promoter.

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