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

Nup98 and Nup100 are required for posttranscriptional dimethylation of H3K4 in human and yeast cells.

<p>(A) Nup98 knockdown was performed as schematized in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001524#pbio-1001524-g006" target="_blank">Figure 6A</a>, and cells were cultured without IFN-γ (uninduced, black), with IFN-γ for 24 h (induced, dark grey), or with IFN-γ for 24 h and then without IFN-γ for 48 h (48 h post, light grey). Cells were fixed and ChIP was performed against H3K4me2. Enrichment of indicated promoters was quantified relative to the input fraction using qPCR. (B) Western blots against Nup107 and GAPDH of lysates from cells treated with siRNA pools against <i>NUP107</i> as schematized in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001524#pbio-1001524-g006" target="_blank">Figure 6A</a>. (C) ChIP against H3K4me2 from cells knocked down for Nup107, quantified as in panel A. (D) Wild-type and <i>nup100</i>Δ cells grown in repressing (+inositol, black), activating (−inositol, dark grey), and recently repressed conditions (−ino→+ino 3 h, light grey) were subjected to ChIP against H3K4me2. Enrichment of the <i>INO1</i> promoter and the <i>PRM1</i> coding sequence were quantified by qPCR. (E) Wild-type and <i>set3</i>Δ mutant strains grown in repressing, activating, and recently repressed conditions were subjected to ChIP against RNAPII. Recovery of the <i>INO1</i> promoter or the <i>GAL1</i> promoter was quantified by qPCR. For all panels, error bars represent the standard error of the mean for three experiments.</p

Many human genes exhibit IFN-γ memory.

<p>(A) The expression change of 664 IFN-γ-inducible genes during activation and reactivation. Shown is the log₂ change in expression from Agilent 128×135K expression microarrays, relative to 0 h (scale below). These genes were subjected to <i>k</i> means clustering to identify three distinct clusters. Cluster 3 includes <i>HLA-DRA</i> (arrow). (B–D) The average change in expression, relative to time = 0, of genes from each cluster during activation and reactivation. (E) Cells were treated as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001524#pbio-1001524-g002" target="_blank">Figure 2B</a>, fixed, and ChIP was performed with Nup98 antisera. The recovery of the promoters and coding sequences of candidate genes from Clusters 1 (<i>HLA-DQB1</i>) and 3 (<i>HLA-DRA</i>, <i>HLA-DPB1</i>, and <i>OAS2</i>) was quantified relative to input by qPCR. Error bars represent the standard error of the mean from three experiments.</p

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

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

Nup98 is necessary for IFN-γ memory.

<p>(A) Schematic of Nup98 knockdown strategy: on the 2 d prior to IFN-γ treatment, cells were serially transfected for 6 h with pools of either scrambled siRNA or <i>NUP98</i> siRNA. (B) Lysates from cells treated as shown in (A) was subjected to Western blot. GAPDH was used as a loading control, and the asterisk indicates nonspecific background for Nup98 antibody. (C) ChIP from cells treated as shown in panel A using anti-RNAPII (8WG16). The promoters and coding sequences of <i>HLA-DRA</i> and <i>CIITA</i> were quantified by qPCR. (D–G) Nup98 was knocked down as indicated in panel A, and cells were then subjected to activation and reactivation regimes as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001524#pbio-1001524-g002" target="_blank">Figure 2A</a>. Legend in panel D applies to panels D–G. The mRNA levels for <i>HLA-DRA</i> (D), <i>HLA-DQB1</i> (E), <i>HLA-DPB1</i> (F), and <i>OAS2</i> (G) were quantified by RT qPCR during activation and reactivation relative to <i>β-ACTIN</i>. For all panels, error bars represent the standard error of the mean for three experiments.</p