The Molecular Basis Of G9A-mediated H3K9Methylation In Autophagy Regulation

Dysregulation of autophagy has been implicated in a wide range of human diseases including cancer. It is generally thought that autophagy functions as a suppressor at the early stages of tumor pathogenesis by eliminating pre-cancerous cells, whereas during tumor progression, autophagy promotes tumor growth and survival under nutrient-limiting and/or hypoxic conditions. Furthermore, it has been shown that induction of autophagy is a mechanism for protecting tumor cells against irradiation and chemotherapy. The proposed research is aimed at a better understanding of the molecular basis of autophagy regulation to address the central question how to precisely control autophagy activation or inhibition for cancer prevention and therapy. Also importantly, our investigation will provide the molecular rationale and the means for targeting histone H3lysine9(H3K9) methyltransferases for the control of autophagic activity in cancer cells.We recently found that small molecule inhibitors of the H3K9methyltransferases G9A and GLP (G9A-like protein), which have a major role in catalyzing H3K9mono-and di-methylation, can trigger robust autophagy in multiple cancer cell lines. This autophagic phenotype can be recapitulated by shRNA-mediated knockdown of G9A expression. The proposed studies will generate new knowledge on the physiological function and molecular mechanism for the previously unknown connection between H3K9methylation and autophagy regulation. Moreover, a molecular understanding of G9A inhibitors in the regulation of autophagic activity will identify a new class of small molecule inhibitors for rapid and user-controlled activation of autophagy, which is anticipated to have broad implications both in experimentally understanding autophagy biology and regulation, and in combating disease or promoting health.Increased activation of the serine-glycine biosynthetic pathway is an integral part of cancer metabolism that drives macromolecule synthesis needed for cell proliferation. Whether this pathway is under epigenetic control is unknown. Here we show that the H3K9methyltransferase G9A is required for maintaining the pathway enzyme genes in an active state marked by H3K9monomethylation and for the transcriptional activation of this pathway in response to serine deprivation. G9A inactivation depletes serine and its downstream metabolites, triggering cell death with autophagy in cancer cell lines of different tissue origins. Higher G9A expression, which is observed in various cancers and is associated with greater mortality in cancer patients, increases serine production and enhances the proliferation and tumorigenicity of cancer cells. These findings identify a G9A-dependent epigenetic program in the control of cancer metabolism, providing a rationale for G9A inhibition as a therapeutic strategy for cancer.BIX01294inhibits tumor cell survivalCells treated with BIX01294showed a significant reduction in both H3K9me1and H3K9me2levels. We treated the human neuroblastoma cell lines BE(2)-C and SHEP1with5μM of BIX01294. Significant levels of cell death were observed following the treatment, as determined by morphology and by trypan blue exclusion assays. The BE(2)-C cells also lost their ability to grow in soft agar. A prominent morphological feature of the neuroblastoma cells treated with BIX01294was the formation of numerous vesicles and vacuoles that were located around the nucleus. This phenotype was observed in all cancer cell lines examined, including U2OS (osteosarcoma), HeLa (cervical), RKO (colon), HCT116(colon), Hep2G (liver), J82(bladder), HEK293(embryonic kidney), and MCF7(breast). Thus, inhibition of tumor cell survival is a general feature of BIX01294.BIX01294induces autophagyBIX01294induces autophagy by electron microscopy for ultrastructural morphology, by immunoblotting for detecting the lipidation of LC3(microtubule-associated protein light chain3) and by immunofluorescence for monitoring the formation of LC3-positive puncta. Electron microscopy revealed numerous double-membraned vacuoles in BIX01294-treated cells that contained fragments of endoplasmic reticulum and other cytoplasmic components. Immunofluorescence staining showed a marked increase in the number of LC3-positive puncta. We performed immunoblot analysis of BIX01294induction of LC3-II in the presence or absence of chloroquine. Chloroquine (CQ) further increased the LC3-II levels in BIX01294-treated cells, indicating that BIX01294increases LC3-II production We independently confirmed the ability of BIX01294to induce autophagy by using wild-type and Atg5-/-mouse embryonic fibroblasts (MEFs). BIX01294was able to induce LC3-positive puncta in wild-type, but not in Atg5-/-MEFs. Together, these findings demonstrate that small molecule inhibitors of G9A are robust inducers of autophagy.G9A is the target of BIX01294action in autophagy inductionWe tested five lentiviral constructs expressing shRNA sequences against different regions of the human G9A gene, and four of them (G9A-shl-4) were highly effective in knockdown of G9A expression, with G9A-sh4showing the highest knockdown efficiency. G9A knockdown, like BIX01294treatment, robustly induced the formation of LC3-positive puncta in cells expressing mCherry-EGFP-LC3B.We transiently transfected293cells with a mCherry-EGFP-LC3B expression construct, along with vector control, G9A, or G9A and G9A-sh4expression constructs. The transfected cells were then treated with BIX01294. In the control cells that only expressed mCherry-EGFP-LC3B, BIX01294treatment induced the formation of LC3B-positive puncta. Overexpression of G9A largely abrogated the ability of BIX01294to induce LC3B-positive puncta, which was reversed by co-expression of G9A-sh4. Together, these findings provide strong evidence that G9A is the primary target of BIX01294action in autophagy induction.G9A inhibition or silencing transcriptionally represses serine-glycine biosynthesisWe examined the kinase activity of the mammalian target of rapamycin complex1(mTORC1). Immunoblotting revealed that BIX significantly inhibited mTORC1kinase activity as demonstrated by a marked decrease in the phosphorylation of the ribosome protein S6kinase (S6K), a key mTORCl substrate. This finding suggests that G9A inhibition triggers autophagy by interfering with cell growth or survival signals upstream of mTORC1.We performed microarray profiling to identify potential G9A targets involved in autophagy induction. A total of615BIX-responsive genes (≥±1.50fold, P<0.01) were identified, with302genes being upregulated and313downregulated. Gene Ontology (GO) analysis revealed that among the genes downregulated by BIX, those within the serine-glycine biosynthetic pathway were significantly enriched, including phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase1 (PSAT1), phosphoserine phosphatase (PSPH), and serine hydroxymethyltransferase2(SHMT2). We confirmed the ability of BIX to downregulate mRNA expression of these genes by quantitative reverse-transcription PCR (qRT-PCR) in four different cancer cell lines. Time-course studies revealed significant downregulation of these genes within2and6hr of BIX treatment in U2OS and HeLa cells, respectively, which occurred at least several hours before most of the cells underwent autophagy. We noticed that SHMT1, though downregulated in HeLa, SHEP1and U2OS cells, was activated in BE(2)-C cells following BIX treatment. The significance of this apparently cell type-dependent regulation of SHMT1expression is currently under investigation. We observed a significant decrease in PHGDH protein levels in BIX-treated cells. Similarly, G9A silencing resulted in marked downregulation of the same group of genes at both mRNA and protein levels.Chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) assay revealed that BIX treatment significantly reduced the H3K9me1levels around the transcriptional start sites (TSS) of PHGDH and PSAT1. By contrast, H3K9me2levels in the same regions were either unchanged or significantly increased following BIX treatment. Together, these results indicate that the serine-glycine biosynthetic pathway is under the direct transcriptional control of G9A, primarily through the regulation of H3K9mel levels associated with the pathway enzyme genes.Supplemental serine rescues the cell death phenotype of G9A inhibitionWe investigated the possibility that suppression of this biosynthetic pathway might be a cause of the cell death phenotype induced by G9A inhibition or silencing. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that only serine and glycine levels were significantly reduced in U2OS cells within4hr of BIX treatment, demonstrating that G9A activity is essential for maintaining the intracellular steady-state levels of serine and glycine. We further assessed the activity of this biosynthetic pathway by [U-13C] glucose flux analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS). BIX treatment significantly decreased the incorporation of [U-13C] glucose into3-phosphoserine and serine. Importantly, addition of serine to the culture medium significantly diminished the effect of BIX or G9A silencing on cell proliferation and autophagy in all the cancer cell lines examined. By contrast, supplemental glycine had only a small protective effect on BIX-treated cells, and addition of both serine and glycine was no more effective than adding serine alone. We confirmed these findings with cell-permeable methyl-serine-ester and methyl-glycine-ester. Other individual amino acid supplements all failed to prevent cell death with autophagy induced by BIX, providing further evidence for the specificity of serine action.We generated SHEP1cells overexpressing SHMT1and SHMT2. Their overexpression alone had no significant effect on BIX-induced cell death. However, overexpression of SHMT2, but not SHMT1, synergized with supplemental serine to enhance cell survival and proliferation in the presence of BIX. Collectively, these results indicate that maintaining the production of serine and its downstream metabolites, including5,10-MTHF, is a major mechanism by which G9A sustains cancer cell survival and proliferation.G9A links serine sensing to ribosome biogenesis and cell-cycle progressionAddition of BIX completely abrogated the induction of PHGDH and PSAT1by serine deprivation, supporting a physiological function of G9A in mediating the serine deprivation response.We performed temporal transcriptome profiling of B IX-responsive genes by RNA sequencing (RNA-seq), which revealed distinctive patterns of gene expression in cells that were treated with BIX for6or24hr. GO analysis of the genes downregulated by BIX at6hr revealed significant enrichment of genes within the serine biosynthetic pathway. However, cells treated with BIX for24hr displayed a gene expression pattern characterized by significant downregulation of genes that control cell cycle progression, including cyclins A2and B2, and CDC25C. We confirmed the RNA-seq result by qRT-PCR, which showed sequential downregulation of serine synthesis and cell cycle genes following BIX treatment. We also confirmed the time-dependent downregulation of cyclins A2and B1by immunoblotting. Importantly, we found marked downregulation of genes involved in ribosome biogenesis in cells treated with BIX for24hr compared to those treated for6hr. Collectively, these results suggest a key role of G9A in coupling serine sensing to the transcription of genes that control protein synthesis and cell proliferation.G9A has an oncogenic potential in tumorigenesisWe found that ectopic expression of G9A in SHEP1and U2OS cells significantly increased cell proliferation and the expression of genes that promote cell cycle progression, indicating that high G9A expression alone is sufficient to confer a growth advantage to cancer cells. Moreover, G9A overexpression markedly enhanced the anchorage-independent growth and tumorigenicity of SHEP1and U2OS cells, demonstrating that G9A has transforming potential. Together, our results suggest an oncogenic potential of G9A in cancer development.Activation of the serine-glycine biosynthetic pathway is essential for the oncogenic activity of G9AEctopic expression of G9A in SHEP1and U2OS cells markedly increased the mRNA and protein levels of the pathway enzyme genes. Moreover, ChIP in combination with DNA sequencing (ChIP-seq) revealed a significant increase in H3K9mel levels at these gene loci. We observed no significant change in H3K9me2levels in the same regions. These results suggest that G9A activates the expression of the pathway enzyme genes by increasing the H3K9mel levels associated with their loci. As expected, G9A overexpression significantly increased the intracellular levels of serine and glycine, and the flux of glucose into the serine biosynthetic pathway.We silenced the expression of PHGDH or PSAT1in G9A-overexpressing U2OS cells by shRNA. Knockdown of either PHGDH or PS ATI abolished the ability of G9A to promote cell proliferation in a manner dependent on levels of PHGDH or PSAT1downregulation. In fact, G9A-overexpressing cells with more than90%of PHGDH or PSAT1knockdown failed to proliferate in the absence of supplemental serine. We obtained essentially the same results with G9A-overexpressing SHEP1. Collectively, these data demonstrate that G9A promotes cell proliferation by transcriptionally activating the serine-glycine biosynthetic pathway.