Epigenetic therapy induces transcription of inverted SINEs and ADAR1 dependency.

Cancer therapies that target epigenetic repressors can mediate their effects by activating retroelements within the human genome. Retroelement transcripts can form double-stranded RNA (dsRNA) that activates the MDA5 pattern recognition receptor1-6. This state of viral mimicry leads to loss of cancer cell fitness and stimulates innate and adaptive immune responses7,8. However, the clinical efficacy of epigenetic therapies has been limited. To find targets that would synergize with the viral mimicry response, we sought to identify the immunogenic retroelements that are activated by epigenetic therapies. Here we show that intronic and intergenic SINE elements, specifically inverted-repeat Alus, are the major source of drug-induced immunogenic dsRNA. These inverted-repeat Alus are frequently located downstream of 'orphan' CpG islands9. In mammals, the ADAR1 enzyme targets and destabilizes inverted-repeat Alu dsRNA10, which prevents activation of the MDA5 receptor11. We found that ADAR1 establishes a negative-feedback loop, restricting the viral mimicry response to epigenetic therapy. Depletion of ADAR1 in patient-derived cancer cells potentiates the efficacy of epigenetic therapy, restraining tumour growth and reducing cancer initiation. Therefore, epigenetic therapies trigger viral mimicry by inducing a subset of inverted-repeats Alus, leading to an ADAR1 dependency. Our findings suggest that combining epigenetic therapies with ADAR1 inhibitors represents a promising strategy for cancer treatment.

N-acetylcysteine reverses the decrease of DNA methylation status caused by engineered gold, silicon, and chitosan nanoparticles.

Introduction: Engineered nanoparticles (ENPs) are one of the most widely used types of nanomaterials. Recently, ENPs have been shown to cause cellular damage by inducing ROS (reactive oxygen species) both directly and indirectly, leading to the changes in DNA methylation levels, which is an important epigenetic mechanism. In this study, we investigated the effect of ENP-induced ROS on DNA methylation. Materials and methods: Human embryonic kidney and human keratinocyte (HaCaT) cells were exposed to three different types of ENPs: gold nanoparticles, silicon nanoparticles (SiNPs), and chitosan nanoparticles (CSNPs). We then evaluated the cytotoxicity of the ENPs by measuring cell viability, morphology, cell apoptosis, cell proliferation, cell cycle distribution and ROS levels. Global DNA methylation levels was measured using 5-methylcytosine immunocytochemical staining and HPLC analysis. DNA methylation levels of the transposable elements, long interspersed element-1 (LINE-1) and Alu, were also measured using combined bisulfite restriction analysis technique. DNA methylation levels of the TEs LINE-1 and Alu were also measured using combined bisulfite restriction analysis technique. Results: We found that HaCaT cells that were exposed to SiNPs exhibited increased ROS levels, whereas HaCaT cells that were exposed to SiNPs and CSNPs experienced global and Alu hypomethylation, with no change in LINE-1 being observed in either cell line. The demethylation of Alu in HaCaT cells following exposure to SiNPs and CSNPs was prevented when the cells were pretreated with an antioxidant. Conclusion: The global DNA methylation that is observed in cells exposed to ENPs is associated with methylation of the Alu elements. However, the change in DNA methylation levels following ENP exposure is specific to particular ENP and cell types and independent of ROS, being induced indirectly through disruption of the oxidative defense process.

Effects on prostate cancer cells of targeting RNA polymerase III.

RNA polymerase (pol) III occurs in two forms, containing either the POLR3G subunit or the related paralogue POLR3GL. Whereas POLR3GL is ubiquitous, POLR3G is enriched in undifferentiated cells. Depletion of POLR3G selectively triggers proliferative arrest and differentiation of prostate cancer cells, responses not elicited when POLR3GL is depleted. A small molecule pol III inhibitor can cause POLR3G depletion, induce similar differentiation and suppress proliferation and viability of cancer cells. This response involves control of the fate-determining factor NANOG by small RNAs derived from Alu short interspersed nuclear elements. Tumour initiating activity in vivo can be reduced by transient exposure to the pol III inhibitor. Untransformed prostate cells appear less sensitive than cancer cells to pol III depletion or inhibition, raising the possibility of a therapeutic window.

Infant peripheral blood repetitive element hypomethylation associated with antiretroviral therapy in utero.

The use of combination antiretroviral therapy (cART) to prevent HIV mother-to-child transmission during pregnancy and delivery is generally considered safe. However, vigilant assessment of potential risks of these agents remains warranted. Epigenetic changes including DNA methylation are considered potential mechanisms linking the in utero environment with long-term health outcomes. Few studies have examined the epigenetic effects of prenatal exposure to pharmaceutical agents, including antiretroviral therapies, on children. In this study, we examined the methylation status of the LINE-1 and ALU-Yb8 repetitive elements as markers of global DNA methylation alteration in peripheral blood mononuclear cells obtained from newborns participating in the Pediatric HIV/AIDS Cohort Study SMARTT cohort of HIV-exposed, cART-exposed uninfected infants compared to a historical cohort of HIV-exposed, antiretroviral-unexposed infants from the Women and Infants Transmission Study Cohort. In linear regression models controlling for potential confounders, we found the adjusted mean difference of AluYb8 methylation of the cART-exposed compared to the -unexposed was -0.568 (95% CI: -1.023, -0.149) and for LINE-1 methylation was -1.359 (95% CI: -1.860, -0.857). Among those exposed to cART, subjects treated with atazanavir (ATV), compared to those on other treatments, had less AluYb8 methylation (-0.524, 95% CI: -0.025, -1.024). Overall, these results suggest a small but statistically significant reduction in the methylation of these repetitive elements in an HIV-exposed, cART-exposed cohort compared to an HIV-exposed, cART-unexposed historic cohort. The potential long-term implications of these differences are worthy of further examination.

The potential role of Alu Y in the development of resistance to SN38 (Irinotecan) or oxaliplatin in colorectal cancer.

BACKGROUND: Irinotecan (SN38) and oxaliplatin are chemotherapeutic agents used in the treatment of colorectal cancer. However, the frequent development of resistance to these drugs represents a considerable challenge in the clinic. Alus as retrotransposons comprise 11% of the human genome. Genomic toxicity induced by carcinogens or drugs can reactivate Alus by altering DNA methylation. Whether or not reactivation of Alus occurs in SN38 and oxaliplatin resistance remains unknown. RESULTS: We applied reduced representation bisulfite sequencing (RRBS) to investigate the DNA methylome in SN38 or oxaliplatin resistant colorectal cancer cell line models. Moreover, we extended the RRBS analysis to tumor tissue from 14 patients with colorectal cancer who either did or did not benefit from capecitabine + oxaliplatin treatment. For the clinical samples, we applied a concept of 'DNA methylation entropy' to estimate the diversity of DNA methylation states of the identified resistance phenotype-associated methylation loci observed in the cell line models. We identified different loci being characteristic for the different resistant cell lines. Interestingly, 53% of the identified loci were Alu sequences- especially the Alu Y subfamily. Furthermore, we identified an enrichment of Alu Y sequences that likely results from increased integration of new copies of Alu Y sequence in the drug-resistant cell lines. In the clinical samples, SOX1 and other SOX gene family members were shown to display variable DNA methylation states in their gene regions. The Alu Y sequences showed remarkable variation in DNA methylation states across the clinical samples. CONCLUSION: Our findings imply a crucial role of Alu Y in colorectal cancer drug resistance. Our study underscores the complexity of colorectal cancer aggravated by mobility of Alu elements and stresses the importance of personalized strategies, using a systematic and dynamic view, for effective cancer therapy.

Sole copy of Z2-type human cytidine deaminase APOBEC3H has inhibitory activity against retrotransposons and HIV-1.

Human cytidine deaminase apolipoprotein B mRNA-editing catalytic polypeptide-like 3 (APOBEC3) proteins have been classified as either Z1- or Z2-type cytidine deaminases on the basis of phylogenetic analysis of their catalytic domains. Despite the identification of a number of Z1-type domain-containing cytidine deaminases, only one copy of Z2-type cytidine deaminase has been detected in each of the mammalian species evaluated thus far. Z1-type human APOBEC3 proteins are known to exhibit broad activities against diverse retroelements. However, the potential role of the only human Z2-type cytidine deaminase, APOBEC3H (A3H), in the restriction of retroelements has not yet been fully characterized. Here, we demonstrate that human A3H is a potent inhibitor of non-LTR LINE-1 transposition. Interestingly, it was also as efficient as A3G in inhibiting Alu retrotransposition, despite its poor association with Alu RNA. We have further demonstrated, for the first time, that human APOBEC3DE is also a potent inhibitor of Alu retrotransposition. Variants of A3H have divergent antiviral activities against HIV-1-Vif-deficient viruses. Unlike the anti-HIV-1 cytidine deaminases A3G and A3F, A3H is moderately regulated by interferons. These observations suggest that human Z2-type cytidine deaminase A3H variants have varying intrinsic abilities to restrict retroelements and that various APOBEC3 proteins may have evolved distinct inhibitory mechanisms against retroelements.

K562 cells implicate increased chromatin accessibility in Alu transcriptional activation.

Alu repeats in K562 cells are unusually hypomethylated and far more actively transcribed than those in other human cell lines and somatic tissues. Also, the level of Alu RNA in K562 cells is relatively insensitive to cell stresses, namely heat shock, adenovirus infection and treatment with cycloheximide, which increase the abundance of Alu RNA in HeLa and 293 cells. Recent advances in understanding the interactions between DNA methylation, transcriptional activation and chromatin conformation reveal reasons for the constitutively high level of Alu expression in K562 cells. Methylation represses transcription of transiently transfected Alu templates in all cell lines tested but cell stresses do not relieve this repression suggesting that they activate Alu transcription through another pathway. A relatively large fraction of the Alus within K562 chromatin is accessible to restriction enzyme cleavage and cell stresses increase the chromatin accessibility of Alus in HeLa and 293 cells. Cell stress evidently activates Alu transcription by rapidly remodeling chromatin to recruit additional templates.