Catastrophic NAD+ Depletion in Activated T Lymphocytes through Nampt Inhibition Reduces Demyelination and Disability in EAE

Nicotinamide phosphoribosyltransferase (Nampt) inhibitors such as FK866 are potent inhibitors of NAD+ synthesis that show promise for the treatment of different forms of cancer. Based on Nampt upregulation in activated T lymphocytes and on preliminary reports of lymphopenia in FK866 treated patients, we have investigated FK866 for its capacity to interfere with T lymphocyte function and survival. Intracellular pyridine nucleotides, ATP, mitochondrial function, viability, proliferation, activation markers and cytokine secretion were assessed in resting and in activated human T lymphocytes. In addition, we used experimental autoimmune encephalomyelitis (EAE) as a model of T-cell mediated autoimmune disease to assess FK866 efficacy in vivo. We show that activated, but not resting, T lymphocytes undergo massive NAD+ depletion upon FK866-mediated Nampt inhibition. As a consequence, impaired proliferation, reduced IFN-γ and TNF-α production, and finally autophagic cell demise result. We demonstrate that upregulation of the NAD+-degrading enzyme poly-(ADP-ribose)-polymerase (PARP) by activated T cells enhances their susceptibility to NAD+ depletion. In addition, we relate defective IFN-γ and TNF-α production in response to FK866 to impaired Sirt6 activity. Finally, we show that FK866 strikingly reduces the neurological damage and the clinical manifestations of EAE. In conclusion, Nampt inhibitors (and possibly Sirt6 inhibitors) could be used to modulate T cell-mediated immune responses and thereby be beneficial in immune-mediated disorders

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

Data from: Set1/COMPASS and Mediator are repurposed to promote epigenetic transcriptional memory

In yeast and humans, previous experiences can lead to epigenetic transcriptional memory: repressed genes that exhibit mitotically heritable changes in chromatin structure and promoter recruitment of poised RNA polymerase II preinitiation complex (RNAPII PIC), which enhances future reactivation. Here, we show that INO1 memory in yeast is initiated by binding of the Sfl1 transcription factor to the cis-acting Memory Recruitment Sequence, targeting INO1 to the nuclear periphery. Memory requires a remodeled form of the Set1/COMPASS methyltransferase lacking Spp1, which dimethylates histone H3 lysine 4 (H3K4me2). H3K4me2 recruits the SET3C complex, which plays an essential role in maintaining this mark. Finally, while active INO1 is associated with Cdk8- Mediator, during memory, Cdk8+ Mediator recruits poised RNAPII PIC lacking the Kin28 CTD kinase. Aspects of this mechanism are generalizable to yeast and conserved in human cells. Thus, COMPASS and Mediator are repurposed to promote epigenetic transcriptional poising by a highly conserved mechanism

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

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

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

yohheiBrickner.fastq

yohheiBrickner.fast