Inducible disruption of Tet genes results in myeloid malignancy, readthrough transcription, and a heterochromatin-to-euchromatin switch

The three mammalian TET dioxygenases oxidize the methyl group of 5-methylcytosine in DNA, and the oxidized methylcytosines are essential intermediates in all known pathways of DNA demethylation. To define the in vivo consequences of complete TET deficiency, we inducibly deleted all three Tet genes in the mouse genome. Tet1/2/3-inducible TKO (iTKO) mice succumbed to acute myeloid leukemia (AML) by 4 to 5 wk. Single-cell RNA sequencing of Tet iTKO bone marrow cells revealed the appearance of new myeloid cell populations characterized by a striking increase in expression of all members of the stefin/cystatin gene cluster on mouse chromosome 16. In patients with AML, high stefin/cystatin gene expression correlates with poor clinical outcomes. Increased expression of the clustered stefin/cystatin genes was associated with a heterochromatin-to-euchromatin compartment switch with readthrough transcription downstream of the clustered stefin/cystatin genes as well as other highly expressed genes, but only minor changes in DNA methylation. Our data highlight roles for TET enzymes that are distinct from their established function in DNA demethylation and instead involve increased transcriptional readthrough and changes in three-dimensional genome organization

NR4A transcription factors limit CAR T cell function in solid tumours

T cells expressing chimeric antigen receptors (CAR T cells) targeting human CD19 (hCD19) have shown clinical efficacy against B cell malignancies(1,2). CAR T cells have been less effective against solid tumours(3-5), in part because they enter a hyporesponsive ('exhausted' or 'dysfunctional') state(6-9) triggered by chronic antigen stimulation and characterized by upregulation of inhibitory receptors and loss of effector function. To investigate the function of CAR T cells in solid tumours, we transferred hCD19-reactive CAR T cells into hCD19(+) tumour-bearing mice. CD8(+)CAR(+) tumour-infiltrating lymphocytes and CD8(+) endogenous tumour-infiltrating lymphocytes expressing the inhibitory receptors PD-1 and TIM3 exhibited similar profiles of gene expression and chromatin accessibility, associated with secondary activation of nuclear receptor transcription factors NR4A1 (also known as NUR77), NR4A2 (NURR1) and NR4A3 (NOR1) by the initiating transcription factor NFAT (nuclear factor of activated T cells)(10-12). CD8(+) T cells from humans with cancer or chronic viral infections(13-15) expressed high levels of NR4A transcription factors and displayed enrichment of NR4A-binding motifs in accessible chromatin regions. CAR T cells lacking all three NR4A transcription factors (Nr4a triple knockout) promoted tumour regression and prolonged the survival of tumour-bearing mice. Nr4a triple knockout CAR tumour-infiltrating lymphocytes displayed phenotypes and gene expression profiles characteristic of CD8(+) effector T cells, and chromatin regions uniquely accessible in Nr4a triple knockout CAR tumour-infiltrating lymphocytes compared to wild type were enriched for binding motifs for NF-kappa B and AP-1, transcription factors involved in activation of T cells. We identify NR4A transcription factors as having an important role in the cell-intrinsic program of T cell hyporesponsiveness and point to NR4A inhibition as a promising strategy for cancer immunotherapy.

Ubiquitin-Mediated Response to Microsporidia and Virus Infection in <i>C. elegans</i>

<div><p>Microsporidia comprise a phylum of over 1400 species of obligate intracellular pathogens that can infect almost all animals, but little is known about the host response to these parasites. Here we use the whole-animal host <i>C. elegans</i> to show an <i>in vivo</i> role for ubiquitin-mediated response to the microsporidian species <i>Nematocida parisii</i>, as well to the Orsay virus, another natural intracellular pathogen of <i>C. elegans</i>. We analyze gene expression of <i>C. elegans</i> in response to <i>N. parisii</i>, and find that it is similar to response to viral infection. Notably, we find an upregulation of SCF ubiquitin ligase components, such as the cullin ortholog <i>cul-6</i>, which we show is important for ubiquitin targeting of <i>N. parisii</i> cells in the intestine. We show that ubiquitylation components, the proteasome, and the autophagy pathway are all important for defense against <i>N. parisii</i> infection. We also find that SCF ligase components like <i>cul-6</i> promote defense against viral infection, where they have a more robust role than against <i>N. parisii</i> infection. This difference may be due to suppression of the host ubiquitylation system by <i>N. parisii</i>: when <i>N. parisii</i> is crippled by anti-microsporidia drugs, the host can more effectively target pathogen cells for ubiquitylation. Intriguingly, inhibition of the ubiquitin-proteasome system (UPS) increases expression of infection-upregulated SCF ligase components, indicating that a trigger for transcriptional response to intracellular infection by <i>N. parisii</i> and virus may be perturbation of the UPS. Altogether, our results demonstrate an <i>in vivo</i> role for ubiquitin-mediated defense against microsporidian and viral infections in <i>C. elegans</i>.</p></div

The SCF ligases, UPS and autophagy limit the growth of <i>N. parisii</i> in the <i>C. elegans</i> intestine.

<p>A) Fluorescence and bright field images demonstrating FISH staining with a probe against <i>N. parisii</i> rRNA used to quantify pathogen load in the <i>C. elegans</i> intestine following 24 hours of infection with <i>N. parisii</i>. Scale bar  = 100 µm. B–F) Quantification of pathogen load (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004200#s4" target="_blank">Materials and Methods</a>) in nematodes treated with RNAi against SCF ligase components (B), against ubiquitin (<i>ubq-2</i>) and two components of the proteasome (<i>pas-5</i> and <i>rpn-2</i>) (C), against <i>ubq-2</i>, +/− fumagillin (D), against <i>ubq-2</i>, +/− FUdR (E), against autophagy components (F), and the <i>C. elegans</i> TOR ortholog <i>let-363</i> (G). Pathogen area occupying each RNAi-treated animal was normalized to mean L4440 control values. The number of animals analyzed for each condition (n) is indicated. Mean +/− SEM is shown for all analyzed animals (data for B, C, F are from three independent experiments, data for D, G are from two independent experiments and data for E are from one experiment). Each independent experiment was comprised of two separate populations of animals. Statistical significance was assessed using a one-way ANOVA with Dunnett's Multiple Comparisons Test for B, C, and F, with Bonferroni Multiple Comparison Test for D and E, and with student's t-test for G (***<i>p</i><0.001, **<i>p</i><0.01, *<i>p</i><0.05).</p

Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Pfam domain (PF) enrichment analysis of <i>C. elegans</i> genes upreguated by <i>N. parisii</i> infection.

<p>GO term, KEGG pathway, and Pfam domain enrichment was analyzed using online DAVID Bioinformatics Resources 6.7. To eliminate redundancy, each term had at least 30% of associated genes not associated with any other term with a more significant P-value. *For each gene group the number of genes included in the analysis out of the total differentially expressed genes is indicated.</p

Model for SCF E3 ligases and ubiquitin-mediated responses to intracellular infection in <i>C. elegans</i>.

<p>Intracellular microsporidia or viral infection triggers the expression of SCF ligase components in <i>C. elegans</i>, including a large number of F-box genes, the cullin <i>cul-6</i>, and Skp1-related genes, <i>skr-3</i>, <i>-4</i>, and <i>-5</i>. Due to the modularity of the SCF ligase complex, many SCF ligases with vast substrate recognition potential may be formed, which could recognize pathogen-derived proteins or host proteins. Ubiquitylation of substrates leads to their degradation by the proteasome or by autophagy, with large substrates such as microsporidia cells, potentially viral particles, and protein aggregates (not shown), targeted by autophagy, and individual pathogen or host proteins by the proteasome. <i>N. parisii</i> parasite cells may be able to suppress or evade ubiquitylation. Both intracellular infection and UPS stress can induce SCF ligase components, and greater demand on the UPS during intracellular infection may contribute to upregulation of SCF ligase components. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004200#s3" target="_blank">Discussion</a> for more details.</p

Ubiquitin-mediated host response and defense against Orsay viral infection in <i>C. elegans</i>.

<p>A) Viral pathogen load in nematodes treated with RNAi against the SCF ligase components <i>skr-3</i>, <i>skr-4, skr-5</i>, and <i>cul-6</i> compared to vector control RNAi (L4440), as assessed by qRT-PCR for viral transcript. Mean +/− SEM of three independent experiments shown. B) Viral pathogen load in nematodes treated with RNAi against <i>ubq-2</i>, <i>pas-5</i> and <i>rpn-2</i> analyzed as above. Mean +/− SEM of three independent experiments shown. C) Intestines of <i>F26F2.1p::gfp</i> transgenic animals were stained with the FK2 antibody against conjugated ubiquitin (red), and DAPI for DNA (blue). Intestines outlined with white dotted line. Animals infected with virus have increased ubiquitin clustering in some intestinal cells compared to uninfected animals (arrowhead), and also express the GFP reporter. Scale bar  = 20 µm. ** p<0.01.</p

<i>N. parisii</i> cells are almost never targeted by ubiquitin later during infection, but ubiquitin forms clusters that accumulate in the <i>C. elegans</i> intestine.

<p>A,B) <i>C. elegans</i> intestines stained an anti-conjugated-ubiquitin antibody FK2 (green), and DAPI for DNA (blue): Panel B also includes a FISH probe against <i>N. parisii</i> rRNA (red). A) Sections of uninfected and <i>N. parisii</i>-infected <i>C. elegans</i> intestines. In the infected intestine an <i>N. parisii</i> spore labeled with the anti-conjugated-ubiquitin antibody is enlarged in box inset in upper left, and meront (arrowhead), and host nucleus (N) are indicated. Scale bar  = 10 µm. B) Sections of uninfected and <i>N. parisii</i>-infected <i>C. elegans</i> intestines (30 hpi) shown with ubiquitin cluster (arrow) and ubiquitin staining within an <i>N. parisii</i> meront (arrowhead) indicated. Scale bar  = 10 µm. C) Intestines of uninfected and infected animals expressing an intestinal GFP::ubiquitin transgene (48 hpi, grown at 20°C to prevent construct aggregation) are shown. Small GFP::ubiquitin aggregates are sometimes observed in uninfected animals (arrow). Scale bar  = 20 µm. D) Enlarged portion of box in panel C. Oblong <i>N. parisii</i> meronts are visible through the absence of green (arrowhead) and ubiquitin clusters associating with the meronts (arrow) are indicated. Scale bar  = 10 µm. E) Animals expressing the intestinal GFP::ubiquitin construct were infected with <i>N. parisii</i> and, together with control uninfected animals, fixed at the indicated times. Fixed animals were stained with a FISH probe against <i>N. parisii</i> rRNA to mark the infection and their intestinal cells were inspected for visible GFP::ubiquitin aggregates (30 transgenic animals were inspected per timepoint and condition). F) Animals expressing the intestinal control GFP::ubiquitinΔGG construct were treated and analyzed as in E.</p

<i>N. parisii</i> cells are targeted by host ubiquitin early during infection.

<p>A–C) <i>C. elegans</i> intestines stained with a FISH probe against <i>N. parisii</i> rRNA (red), and DAPI for DNA (blue). A section of the image outlined by a dotted square is enlarged on the right and shows both an <i>N. parisii</i> parasite cell that colocalizes with ubiquitin (arrow), and one that does not (arrowhead). A) Animals were stained with an anti-conjugated-ubiquitin antibody, FK2 (green), and B, C) Transgenic <i>C. elegans</i> intestines expressing wild-type (B) or conjugation-defective mutant (C) GFP::ubiquitin (green). For A–C, scale bar  = 10 µm in main images and 2 µm in enlarged sections. D) Quantification of parasite cell colocalization at 12 hpi (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004200#s4" target="_blank">Materials and Methods</a> for timepoint information) with FK2 antibody in the presence of increasing doses of fumagillin. Difference is significant: chi-squared test, p<6.3E-28, all comparisons. E) Quantification of parasite cell colocalization at 15 hpi with wild-type or mutant GFP::ubiquitin in the presence of increasing doses of fumagillin. Mean +/− SEM of two independent experiments is shown. F) Quantification of parasite cell colocalization at 15 hpi with wild-type GFP::ubiquitin following knockdown of <i>cul-6</i> RNAi compared to L4440 vector control. Targeting of ubiquitin to parasite cells was less robust and more variable in animals feeding on HT115 RNAi bacteria compared to OP50-1 <i>E. coli</i>, ranging from 10.6% to 2.2% in control animals, and 2.1% to 1.8% in <i>cul-6</i> RNAi treated animals and the data presented are normalized for the average level of targeting in each independent experiment. Mean +/− SEM of three independent experiments is shown. Number of individual parasite cells assessed for colocalization with ubiquitin is indicated.</p