Extensive Cooperation of Immune Master Regulators IRF3 and NFκB in RNA Pol II Recruitment and Pause Release in Human Innate Antiviral Transcription

Transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor κB (NFκB) are activated by external stimuli, including virus infection, to translocate to the nucleus and bind genomic targets important for immunity and inflammation. To investigate RNA polymerase II (Pol II) recruitment and elongation in the human antiviral gene regulatory network, a comprehensive genome-wide analysis was conducted during the initial phase of virus infection. Results reveal extensive integration of IRF3 and NFκB with Pol II and associated machinery and implicate partners for antiviral transcription. Analysis indicates that both de novo polymerase recruitment and stimulated release of paused polymerase work together to control virus-induced gene activation. In addition to known messenger-RNA-encoding loci, IRF3 and NFκB stimulate transcription at regions not previously associated with antiviral transcription, including abundant unannotated loci that encode novel virus-inducible RNAs (nviRNAs). These nviRNAs are widely induced by virus infections in diverse cell types and represent a previously overlooked cellular response to virus infection

A conserved role for human Nup98 in altering chromatin structure and promoting epigenetic transcriptional memory.

The interaction of nuclear pore proteins (Nups) with active genes can promote their transcription. In yeast, some inducible genes interact with the nuclear pore complex both when active and for several generations after being repressed, a phenomenon called epigenetic transcriptional memory. This interaction promotes future reactivation and requires Nup100, a homologue of human Nup98. A similar phenomenon occurs in human cells; for at least four generations after treatment with interferon gamma (IFN-γ), many IFN-γ-inducible genes are induced more rapidly and more strongly than in cells that have not previously been exposed to IFN-γ. In both yeast and human cells, the recently expressed promoters of genes with memory exhibit persistent dimethylation of histone H3 lysine 4 (H3K4me2) and physically interact with Nups and a poised form of RNA polymerase II. However, in human cells, unlike yeast, these interactions occur in the nucleoplasm. In human cells transiently depleted of Nup98 or yeast cells lacking Nup100, transcriptional memory is lost; RNA polymerase II does not remain associated with promoters, H3K4me2 is lost, and the rate of transcriptional reactivation is reduced. These results suggest that Nup100/Nup98 binding to recently expressed promoters plays a conserved role in promoting epigenetic transcriptional memory

A subset of PIC components interacts with the <i>INO1</i> promoter after repression.

<p>(A) TAP-tagged strains were grown under long-term repressing (+inositol, black bars), activating (−inositol, dark grey bars), or recently repressed (−ino→+ino 3 h, light grey bars) conditions and processed for chromatin immunoprecipitation (ChIP). The recovery of the <i>INO1</i> promoter was quantified by qPCR relative to input. Individual tagged subunits and the PIC complex component are indicated. (B) Cells with (grey bars) or without (black bars) the MRS inserted beside <i>URA3 </i><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001524#pbio.1001524-Light1" target="_blank">[18]</a>, grown in recently repressed conditions, were fixed and subjected to ChIP using anti-RNAPII (8WG16). The recovery of the <i>INO1</i> promoter or the indicated loci was quantified by qPCR relative to input. For all panels, error bars represent standard error of the mean from three experiments.</p

Human <i>HLA-DRA</i> exhibits epigenetic transcriptional memory.

<p>(A, Top) Schematic of activation and reactivation time courses. For activation, HeLa cells were mock treated for 72 h and then treated with IFN-γ. For reactivation, cells were first treated with IFN-γ for 24 h, washed and split into fresh medium, cultured for 48 h without IFN-γ, and then treated again with IFN-γ. (Bottom) RT qPCR on RNA harvested from cells at the indicated times during activation and reactivation. The levels of <i>HLA-DRA</i> mRNA were quantified relative to <i>β</i>-<i>ACTIN</i>. The positions of all qPCR products for human genes are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001524#pbio.1001524.s003" target="_blank">Figure S3</a>. Error bars represent the standard error of the mean from five experiments. (B, Top) Schematic of treatment regime. (Bottom) Cells were fixed and harvested at the indicated times and ChIP was performed using anti-RNAPII (8WG16). The recovery of promoter and coding sequences for <i>HLA-DRA</i>, <i>CIITA</i>, and <i>GAPDH</i> was quantified by qPCR relative to input. (C) Cells were treated as in panel B, fixed, and ChIP was performed using anti-phospho-Ser5 CTD (4h8). The recovery of promoter and coding sequences for <i>HLA-DRA</i>, <i>CIITA</i>, and <i>GAPDH</i> was quantified by qPCR relative to input. For panels B and C, error bars represent standard error of the mean from three experiments.</p

H3K4 dimethylation is necessary for <i>INO1</i> transcriptional memory.

<p>For panels A–F, cells were grown under long-term repressing (+inositol, black bars), activating (−inositol, dark grey bars), or recently repressed (−ino→+ino 3 h, light grey bars) conditions. For panels A, B, D, E, and F, the recovery of the <i>INO1</i> promoter was quantified by qPCR relative to input. For all panels, error bars represent the standard error of the mean from three experiments. Wild-type and <i>mrs</i> mutant strains were fixed and subjected to ChIP using anti-H3K4me3 (A) or anti-H3K4me2 (B). The <i>GAL1-10</i> promoter served as a negative control in panels A and B. (C) <i>INO1</i> peripheral localization was quantified by localizing the LacO array bound to LacI-GFP with respect to the nuclear envelope, stained against Sec63-myc <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001524#pbio.1001524-Brickner3" target="_blank">[91]</a>. The blue, hatched line represents the baseline for peripheral localization in this assay <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001524#pbio.1001524-Brickner1" target="_blank">[12]</a>. Three biological replicates of 30–50 cells were scored. (D) Wild-type, <i>set1</i>Δ, and <i>rad6</i>Δ cells were fixed and subjected to ChIP using anti-RNAPII (8WG16). (E) The MRS or <i>mrs mutant</i> was inserted at <i>URA3 </i><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001524#pbio.1001524-Ahmed1" target="_blank">[15]</a>, and cells were fixed and subjected to ChIP using anti-H3K4me2. The recovery of the <i>INO1</i> promoter or the insertion site at <i>URA3</i> was quantified by qPCR relative to input. (F) Wild-type and <i>htz1</i>Δ cells were fixed and subjected to ChIP using anti-H3K4me2.</p