SETD2 regulates chromatin accessibility and transcription to suppress lung tumorigenesis

SETD2, a H3K36 trimethyltransferase, is the most frequently mutated epigenetic modifier in lung adenocarcinoma, with a mutation frequency of approximately 9%. However, how SETD2 loss of function promotes tumorigenesis remains unclear. Using conditional Setd2-KO mice, we demonstrated that Setd2 deficiency accelerated the initiation of KrasG12D-driven lung tumorigenesis, increased tumor burden, and significantly reduced mouse survival. An integrated chromatin accessibility and transcriptome analysis revealed a potentially novel tumor suppressor model of SETD2 in which SETD2 loss activates intronic enhancers to drive oncogenic transcriptional output, including the KRAS transcriptional signature and PRC2-repressed targets, through regulation of chromatin accessibility and histone chaperone recruitment. Importantly, SETD2 loss sensitized KRAS-mutant lung cancer to inhibition of histone chaperones, the FACT complex, or transcriptional elongation both in vitro and in vivo. Overall, our studies not only provide insight into how SETD2 loss shapes the epigenetic and transcriptional landscape to promote tumorigenesis, but they also identify potential therapeutic strategies for SETD2 mutant cancers

Protective Coupling of Mitochondrial Function and Protein Synthesis via the eIF2α Kinase GCN-2

Cells respond to defects in mitochondrial function by activating signaling pathways that restore homeostasis. The mitochondrial peptide exporter HAF-1 and the bZip transcription factor ATFS-1 represent one stress response pathway that regulates the transcription of mitochondrial chaperone genes during mitochondrial dysfunction. Here, we report that GCN-2, an eIF2α kinase that modulates cytosolic protein synthesis, functions in a complementary pathway to that of HAF-1 and ATFS-1. During mitochondrial dysfunction, GCN-2–dependent eIF2α phosphorylation is required for development as well as the lifespan extension observed in Caenorhabditis elegans. Reactive oxygen species (ROS) generated from dysfunctional mitochondria are required for GCN-2–dependent eIF2α phosphorylation but not ATFS-1 activation. Simultaneous deletion of ATFS-1 and GCN-2 compounds the developmental defects associated with mitochondrial stress, while stressed animals lacking GCN-2 display a greater dependence on ATFS-1 and stronger induction of mitochondrial chaperone genes. These findings are consistent with translational control and stress-dependent chaperone induction acting in complementary arms of the UPRmt

SWI/SNF tumor suppressor gene PBRM1/BAF180 in human clear cell kidney cancer

Mutations within chromatin modulating protein complexes have dominated the novel cancer gene landscape. However, little is known about how individual aberrations contribute to cancer formation. A novel Pbrm1 kidney cancer mouse model examining the role of Pbrm1 provides much needed clue concerning how SWI/SNF complexes might function as tumor suppressors

The C. elegans CCAAT-Enhancer-Binding Protein Gamma Is Required for Surveillance Immunity.

Pathogens attack host cells by deploying toxins that perturb core host processes. Recent findings from the nematode C. elegans and other metazoans indicate that surveillance or "effector-triggered" pathways monitor functioning of these core processes and mount protective responses when they are perturbed. Despite a growing number of examples of surveillance immunity, the signaling components remain poorly defined. Here, we show that CEBP-2, the C. elegans ortholog of mammalian CCAAT-enhancer-binding protein gamma, is a key player in surveillance immunity. We show that CEBP-2 acts together with the bZIP transcription factor ZIP-2 in the protective response to translational block by P. aeruginosa Exotoxin A as well as perturbations of other processes. CEBP-2 serves to limit pathogen burden, promote survival upon P. aeruginosa infection, and also promote survival upon Exotoxin A exposure. These findings may have broad implications for the mechanisms by which animals sense pathogenic attack and mount protective responses

Knockdown of GCN-2 and GSP-1 Modulates eIF2α Phosphorylation Status and Mitochondrial Protein Homeostasis.

<p>(A) Fluorescent photomicrographs of <i>hsp-4_pr::gfp</i> reporter animals raised on vector(RNAi), <i>gcn-2</i>(RNAi) or <i>pek-1</i>(RNAi). Worms were hatched on the individual RNAi plates and maintained at 20°C (upper panels) or subjected to heat shock at 30°C (3 hours) to induce ER stress (lower panels) at the L4 developmental stage. (B) Comparison of the amino acid sequence surrounding the conserved serine residue of eIF2α that is phosphorylated by the eIF2α kinases including GCN-2. (C) Immunoblot of wild-type worm lysates untreated or treated with calf intestinal phosphatase (CIP) and probed with an antibody specific to the phosphorylated form of eIF2α. The endogenous ER protein HDEL was detected with a monoclonal antibody (lower panel) and serves as a loading control. (D) Immunoblot of phosphorylated eIF2α from wild-type, <i>gcn-2(ok871)</i> and <i>gcn-2(ok871);pek-1(zcdf2)</i> animals. The anti-eIF2α and anti-HDEL immunoblots serve as loading controls. (E) Immunoblot of phosphorylated eIF2α from wild-type, <i>gcn-2(ok871)</i>, <i>pek-1(zcdf2)</i> and <i>gcn-2(ok871);pek-1(zcdf2)</i> animals raised on vector or <i>gsp-1</i>(RNAi). The anti-HDEL immunoblot serves as a loading control. Animals were raised from eggs on vector or <i>gsp-1</i>(RNAi) and harvested at the L4 stage.</p

Phosphorylation of eIF2α during Mitochondrial Stress Requires GCN-2.

<p>(A) Immunoblot of phospho-eIF2α from wild-type, <i>clk-1(qm30)</i> and <i>clk-1(qm30);gcn-2(ok871)</i> animals fed vector or <i>gsp-1</i>(RNAi). The total eIF2α and anti-HDEL immunoblots serve as loading controls. Synchronized animals were raised from eggs and harvested at the L4 stage. (B) Immunoblot of phospho-eIF2α from wild-type, <i>isp-1(qm150)</i> and <i>isp-1(qm150);gcn-2(ok871)</i> animals fed vector or <i>gsp-1</i>(RNAi). The anti-HDEL immunoblot serves as a loading control. Synchronized animals were raised from eggs on the indicated RNAi plates and harvested at the L4 stage. (C) Immunoblot of phospho-eIF2α from wild-type, <i>clk-1(qm30)</i>, <i>clk-1(qm30)</i>;<i>gcn-2(ok871)</i> or <i>clk-1(qm30)</i>;<i>pek-1(zcdf2)</i> worms. The anti-HDEL immunoblot serves as a loading control. Synchronized animals were raised from eggs on vector(RNAi) plates and harvested at the L4 stage.</p

Identification of Kinases and Phosphatases That Affect Protein Synthesis Impact UPR^mt Activation.

<p>(A) Phosphatase and kinases whose knockdown by RNAi either increased or decreased <i>hsp-60_pr::gfp</i> expression in <i>clk-1(qm30)</i> mutant worms. (B) Fluorescent photomicrographs of <i>hsp-60_pr::gfp</i> expression in wild-type and <i>clk-1(qm30)</i> animals raised on vector, <i>gcn-2</i> or <i>gsp-1</i>(RNAi).</p

GCN-2 Acts in a Complementary Protective Pathway to that of ATFS-1 and the Induction of Mitochondrial Chaperone Genes.

<p>(A) Immunoblot of phosphorylated eIF2α from wild-type, <i>clk-1(qm30)</i>, <i>clk-1(qm30);gcn-2(ok871)</i>, <i>clk-1(qm30);haf-1(ok705)</i> animals fed vector(RNAi) and <i>clk-1(qm30)</i> animals fed <i>atfs-1</i>(RNAi). The anti-HDEL immunoblot serves as a loading control. Worms were synchronized and raised from eggs on the indicated RNAi plate and harvested at the L4 stage. (B) Quantification of developmental rates of <i>gcn-2(ok871)</i>, <i>atfs-1(tm4525)</i> and <i>atfs-1(tm4525);gcn-2(ok871)</i> animals. Synchronized worms were raised from eggs and scored as percent of total animals on day 3. (C) Quantification of developmental rates of <i>atfs-1(tm4525)</i> and <i>atfs-1(tm4525);gcn-2(ok871)</i> animals raised on vector(RNAi) or <i>spg-7</i>(RNAi). Synchronized worms were raised from eggs and scored as percent of total animals on day 3. (D) Quantification of developmental rates of <i>clk-1(qm30)</i> and <i>clk-1(qm30);gcn-2(ok871)</i> animals raised on vector(RNAi) or <i>atfs-1</i>(RNAi). Synchronized worms were raised from eggs and scored as percent of total animals on day 6. (E) Lifespan analysis of wild-type (median lifespan 20.0 days) and <i>atfs-1(tm4525);gcn-2(ok871)</i> animals (median lifespan 18.0 days); p = 0.3230, log-rank test. (F) Scheme of the hypothesized relationship of the two branches of the UPR^mt where HAF-1 and ATFS-1 regulate mitochondrial chaperone gene induction and GCN-2 phosphorylates eIF2α to attenuate protein translation in response to mitochondrial stress.</p

ATFS-1 Is Required for Mitochondrial Chaperone Induction and Development during Mitochondrial Stress.

<p>(A) Fluorescent photomicrographs of wild-type and <i>atfs-1(tm4525);hsp-60_pr::gfp</i> transgenic worms raised on vector or <i>spg-7</i>(RNAi). (B) Representative fluorescent photomicrographs of <i>hsp-60_pr::gfp</i> transgenic worms harboring the <i>clk-1(qm30)</i> or <i>isp-1(qm150)</i> alleles raised on vector(RNAi). (C) Images of <i>clk-1(qm30)</i> animals raised on vector or <i>atfs-1</i>(RNAi). Worms were plated at the L4 stage, allowed to develop to adulthood and lay eggs for 16 hours. The images were obtained five days after hatching.</p