Combination of novel oncolytic herpesvirus with paclitaxel as an efficient strategy for breast cancer therapy.

BACKGROUND: New strategies are needed to improve the treatment of patients with breast cancer (BC). Oncolytic virotherapy is a promising new tool for cancer treatment but still has a limited overall durable antitumor response. A novel replicable recombinant oncolytic herpes simplex virus type 1 called VG161 has been developed and has demonstrated antitumor effects in several cancers. Here, we explored the efficacy and the antitumor immune response of VG161 cotreatment with paclitaxel (PTX) which as a novel oncolytic viral immunotherapy for BC. METHODS: The antitumor effect of VG161 and PTX was confirmed in a BC xenograft mouse model. The immunostimulatory pathways were tested by RNA-seq and the remodeling of tumor microenvironment was detected by Flow cytometry analysis or Immunohistochemistry. Pulmonary lesions were analyzed by the EMT6-Luc BC model. RESULTS: In this report, we demonstrate that VG161 can significantly represses BC growth and elicit a robust antitumor immune response in a mouse model. The effect is amplified when combined with PTX treatment. The antitumor effect is associated with the infiltration of lymphoid cells, including CD4+ T cells, CD8+ T cells, and NK cells (expressing TNF and IFN-γ), and myeloid cells, including macrophages, myeloid-derived suppressor cells, and dendritic cell cells. Additionally, VG161 cotreatment with PTX showed a significant reduction in BC lung metastasis, which may result from the enhanced CD4+ and CD8+ T cell-mediated responses. CONCLUSIONS: The combination of PTX and VG161 is effective for repressing BC growth by inducing proinflammatory changes in the tumor microenvironment and reducing BC pulmonary metastasis. These data will provide a new strategy and valuable insight for oncolytic virus therapy applications in primary solid or metastatic BC tumors.

Molecular characterization of host-specific biofilm formation in a vertebrate gut symbiont.

Although vertebrates harbor bacterial communities in their gastrointestinal tract whose composition is host-specific, little is known about the mechanisms by which bacterial lineages become selected. The goal of this study was to characterize the ecological processes that mediate host-specificity of the vertebrate gut symbiont Lactobacillus reuteri, and to systematically identify the bacterial factors that are involved. Experiments with monoassociated mice revealed that the ability of L. reuteri to form epithelial biofilms in the mouse forestomach is strictly dependent on the strain's host origin. To unravel the molecular basis for this host-specific biofilm formation, we applied a combination of transcriptome analysis and comparative genomics and identified eleven genes of L. reuteri 100-23 that were predicted to play a role. We then determined expression and importance of these genes during in vivo biofilm formation in monoassociated mice. This analysis revealed that six of the genes were upregulated in vivo, and that genes encoding for proteins involved in epithelial adherence, specialized protein transport, cell aggregation, environmental sensing, and cell lysis contributed to biofilm formation. Inactivation of a serine-rich surface adhesin with a devoted transport system (the SecA2-SecY2 pathway) completely abrogated biofilm formation, indicating that initial adhesion represented the most significant step in biofilm formation, likely conferring host specificity. In summary, this study established that the epithelial selection of bacterial symbionts in the vertebrate gut can be both specific and highly efficient, resulting in biofilms that are exclusively formed by the coevolved strains, and it allowed insight into the bacterial effectors of this process.

The functional role of L-fucose on dendritic cell function and polarization.

Despite significant advances in the development and refinement of immunotherapies administered to combat cancer over the past decades, a number of barriers continue to limit their efficacy. One significant clinical barrier is the inability to mount initial immune responses towards the tumor. As dendritic cells are central initiators of immune responses in the body, the elucidation of mechanisms that can be therapeutically leveraged to enhance their functions to drive anti-tumor immune responses is urgently needed. Here, we report that the dietary sugar L-fucose can be used to enhance the immunostimulatory activity of dendritic cells (DCs). L-fucose polarizes immature myeloid cells towards specific DC subsets, specifically cDC1 and moDC subsets. In vitro, L-fucose treatment enhances antigen uptake and processing of DCs. Furthermore, our data suggests that L-fucose-treated DCs increase stimulation of T cell populations. Consistent with our functional assays, single-cell RNA sequencing of intratumoral DCs from melanoma- and breast tumor-bearing mice confirmed transcriptional regulation and antigen processing as pathways that are significantly altered by dietary L-fucose. Together, this study provides the first evidence of the ability of L-fucose to bolster DC functionality and provides rational to further investigate how L-fucose can be used to leverage DC function in order to enhance current immunotherapy.

Study on the Damage Model of Non-Persistent Jointed Rock Mass under the Coupling of Freeze-Thaw and Shear.

Considering that a jointed rock mass in a cold area is often affected by periodic freeze-thaw cycles and shear failure, definitions for the mesoscopic and macroscopic damage to a jointed rock mass under the coupling of freeze-thaw and shear are proposed, and the damage mechanism is verified according to experimental results. The results show that: (1) the jointed rock specimens increase macro-joints and meso-defects, the mechanical properties deteriorate significantly under freeze-thaw cycles, and the damage degree becomes more and more significant with the increases in freeze-thaw cycles and joint persistency. (2) When the number of freeze-thaw cycles is constant, the total damage variable value gradually increases with the increase in joint persistency. The damage variable difference in specimens with different persistency is distinct, which is gradually reduced in the later cycles, indicating a weakening influence of persistency on the total damage variable. (3) The shear resistance of non-persistent jointed rock mass in a cold area is determined by the coupling effect of meso-damage and frost heaving macro-damage. The coupling damage variable can accurately describe the damage variation law of jointed rock mass under freeze-thaw cycles and shear load.

Protocol optimization of a targeted sequencing panel for genomic profiling of bronchoalveolar lavage fluid in lung cancer.

INTRODUCTION: We investigated a commercially available sequencing panel to study the effect of sequencing depth, variant calling strategy, and targeted sequencing region on identifying tumor-derived variants in cell-free bronchoalveolar lavage (cfBAL) DNA compared with plasma cfDNA. METHODS: Sequencing was performed at low or high coverage using two filtering algorithms to identify tumor variants on two panels targeting 77 and 197 genes respectively. RESULTS: One hundred and four sequencing files from 40 matched DNA samples of cfBAL, plasma, germline leukocytes, and archival tumor specimens in 10 patients with early-stage lung cancer were analyzed. By low-coverage sequencing, tumor-derived cfBAL variants were detected in 5/10 patients (50%) compared with 2/10 (20%) for plasma. High-coverage sequencing did not affect the number of tumor-derived variants detected in either biospecimen type. Accounting for germline mutations eliminated false-positive plasma calls regardless of coverage (0/10 patients with tumor-derived variants identified) and increased the number of cfBAL calls (5/10 patients with tumor-derived variants identified). These results were not affected by the number of targeted genes.

Protocol for the isolation of CD8+ tumor-infiltrating lymphocytes from human tumors and their characterization by single-cell immune profiling and multiome.

Understanding the heterogenicity of tumor-infiltraing lymphocyte (TIL) populations and the immunobiology in human cancer is a key to establish efficient immunotherapies. Here, we have established a protocol for the characterization of CD8+ TILs in tumors by single-cell RNA-seq paired to VDJ profiling and chromatin structure including dissociation of tumor biopsies. We have also provided guidance for subsequent fluorescence-activated cell sorting (FACS), single-cell encapsulation, bioinformatics analysis, and troubleshooting. For complete details on the use and execution of this protocol, please refer to Anadon et al. (2022).

Genomic and Single-Cell Landscape Reveals Novel Drivers and Therapeutic Vulnerabilities of Transformed Cutaneous T-cell Lymphoma.

ABSTRACT: Cutaneous T-cell lymphoma (CTCL) is a rare cancer of skin-homing T cells. A subgroup of patients develops large cell transformation with rapid progression to an aggressive lymphoma. Here, we investigated the transformed CTCL (tCTCL) tumor ecosystem using integrative multiomics spanning whole-exome sequencing (WES), single-cell RNA sequencing, and immune profiling in a unique cohort of 56 patients. WES of 70 skin biopsies showed high tumor mutation burden, UV signatures that are prognostic for survival, exome-based driver events, and most recurrently mutated pathways in tCTCL. Single-cell profiling of 16 tCTCL skin biopsies identified a core oncogenic program with metabolic reprogramming toward oxidative phosphorylation (OXPHOS), cellular plasticity, upregulation of MYC and E2F activities, and downregulation of MHC I suggestive of immune escape. Pharmacologic perturbation using OXPHOS and MYC inhibitors demonstrated potent antitumor activities, whereas immune profiling provided in situ evidence of intercellular communications between malignant T cells expressing macrophage migration inhibitory factor and macrophages and B cells expressing CD74. SIGNIFICANCE: Our study contributes a key resource to the community with the largest collection of tCTCL biopsies that are difficult to obtain. The multiomics data herein provide the first comprehensive compendium of genomic alterations in tCTCL and identify potential prognostic signatures and novel therapeutic targets for an incurable T-cell lymphoma. This article is highlighted in the In This Issue feature, p. 1171.

Single-Cell Characterization of the Immune Microenvironment of Melanoma Brain and Leptomeningeal Metastases.

PURPOSE: Melanoma brain metastases (MBM) and leptomeningeal melanoma metastases (LMM) are two different manifestations of melanoma CNS metastasis. Here, we used single-cell RNA sequencing (scRNA-seq) to define the immune landscape of MBM, LMM, and melanoma skin metastases. EXPERIMENTAL DESIGN: scRNA-seq was undertaken on 43 patient specimens, including 8 skin metastases, 14 MBM, and 19 serial LMM specimens. Detailed cell type curation was performed, the immune landscapes were mapped, and key results were validated by IHC and flow cytometry. Association analyses were undertaken to identify immune cell subsets correlated with overall survival. RESULTS: The LMM microenvironment was characterized by an immune-suppressed T-cell landscape distinct from that of brain and skin metastases. An LMM patient with long-term survival demonstrated an immune repertoire distinct from that of poor survivors and more similar to normal cerebrospinal fluid (CSF). Upon response to PD-1 therapy, this extreme responder showed increased levels of T cells and dendritic cells in their CSF, whereas poor survivors showed little improvement in their T-cell responses. In MBM patients, therapy led to increased immune infiltrate, with similar T-cell transcriptional diversity noted between skin metastases and MBM. A correlation analysis across the entire immune landscape identified the presence of a rare population of dendritic cells (DC3) that was associated with increased overall survival and positively regulated the immune environment through modulation of activated T cells and MHC expression. CONCLUSIONS: Our study provides the first atlas of two distinct sites of melanoma CNS metastases and defines the immune cell landscape that underlies the biology of this devastating disease.

Managing a Large-Scale Multiomics Project: A Team Science Case Study in Proteogenomics.

Highly collaborative scientists are often called on to extend their expertise to different types of projects and to expand the scope and scale of projects well beyond their previous experience. For a large-scale project involving "big data" to be successful, several different aspects of the research plan need to be developed and tested, which include but are not limited to the experimental design, sample collection, sample preparation, metadata recording, technical capability, data acquisition, approaches for data analysis, methods for integration of different data types, recruitment of additional expertise as needed to guide the project, and strategies for clear communication throughout the project. To capture this process, we describe an example project in proteogenomics that built on our collective expertise and experience. Key steps included definition of hypotheses, identification of an appropriate clinical cohort, pilot projects to assess feasibility, refinement of experimental designs, and extensive discussions involving the research team throughout the process. The goal of this chapter is to provide the reader with a set of guidelines to support development of other large-scale multiomics projects.

Integrative Analysis of Breast Cancer Cells Reveals an Epithelial-Mesenchymal Transition Role in Adaptation to Acidic Microenvironment.

Early ducts of breast tumors are unequivocally acidic. High rates of glycolysis combined with poor perfusion lead to a congestion of acidic metabolites in the tumor microenvironment, and pre-malignant cells must adapt to this acidosis to thrive. Adaptation to acidosis selects cancer cells that can thrive in harsh conditions and are capable of outgrowing the normal or non-adapted neighbors. This selection is usually accompanied by phenotypic change. Epithelial mesenchymal transition (EMT) is one of the most important switches correlated to malignant tumor cell phenotype and has been shown to be induced by tumor acidosis. New evidence shows that the EMT switch is not a binary system and occurs on a spectrum of transition states. During confirmation of the EMT phenotype, our results demonstrated a partial EMT phenotype in our acid-adapted cell population. Using RNA sequencing and network analysis we found 10 dysregulated network motifs in acid-adapted breast cancer cells playing a role in EMT. Our further integrative analysis of RNA sequencing and SILAC proteomics resulted in recognition of S100B and S100A6 proteins at both the RNA and protein level. Higher expression of S100B and S100A6 was validated in vitro by Immunocytochemistry. We further validated our finding both in vitro and in patients' samples by IHC analysis of Tissue Microarray (TMA). Correlation analysis of S100A6 and LAMP2b as marker of acidosis in each patient from Moffitt TMA approved the acid related role of S100A6 in breast cancer patients. Also, DCIS patients with higher expression of S100A6 showed lower survival compared to lower expression. We propose essential roles of acid adaptation in cancer cells EMT process through S100 proteins such as S100A6 that can be used as therapeutic strategy targeting both acid-adapted and malignant phenotypes.

Genomic instability in regions adjacent to a highly conserved pch prophage in Escherichia coli O157:H7 generates diversity in expression patterns of the LEE pathogenicity island.

The LEE pathogenicity island has been acquired on multiple occasions within the different lineages of enteropathogenic and enterohemorrhagic Escherichia coli. In each lineage, LEE expression is regulated by complex networks of pathways, including core pathways shared by all lineages and lineage-specific pathways. Within the O157:H7 lineage of enterohemorrhagic E. coli, strain-to-strain variation in LEE expression has been observed, implying that expression patterns can diversify even within highly related subpopulations. Using comparative genomics of E. coli O157:H7 subpopulations, we have identified one source of strain-level variation affecting LEE expression. The variation occurs in prophage-dense regions of the genome that lie immediately adjacent to the late regions of the pch prophage carrying pchA, pchB, pchC, and a newly identified pch gene, pchX. Genomic segments extending from the holin S region to the pchA, pchB, pchC, and pchX genes of their respective prophage are highly conserved but are nonetheless embedded within adjacent genomic segments that are extraordinarily variable, termed pch adjacent genomic regions (pch AGR). Despite the remarkable degree of variation, the pattern of variation in pch AGR is highly correlated with the distribution of phylogenetic markers on the backbone of the genome. Quantitative analysis of transcription from the LEE1 promoter further revealed that variation in the pch AGR has substantial effects on absolute levels and patterns of LEE1 transcription. Variation in the pch AGR therefore serves as a mechanism to diversify LEE expression patterns, and the lineage-specific pattern of pch AGR variation could ultimately influence ecological or virulence characteristics of subpopulations within each lineage.

Diet-induced metabolic improvements in a hamster model of hypercholesterolemia are strongly linked to alterations of the gut microbiota.

The mammalian gastrointestinal microbiota exerts a strong influence on host lipid and cholesterol metabolism. In this study, we have characterized the interplay among diet, gut microbial ecology, and cholesterol metabolism in a hamster model of hypercholesterolemia. Previous work in this model had shown that grain sorghum lipid extract (GSL) included in the diet significantly improved the high-density lipoprotein (HDL)/non-HDL cholesterol equilibrium (T. P. Carr, C. L. Weller, V. L. Schlegel, S. L. Cuppett, D. M. Guderian, Jr., and K. R. Johnson, J. Nutr. 135:2236-2240, 2005). Molecular analysis of the hamsters' fecal bacterial populations by pyrosequencing of 16S rRNA tags, PCR-denaturing gradient gel electrophoresis, and Bifidobacterium-specific quantitative real-time PCR revealed that the improvements in cholesterol homeostasis induced through feeding the hamsters GSL were strongly associated with alterations of the gut microbiota. Bifidobacteria, which significantly increased in abundance in hamsters fed GSL, showed a strong positive association with HDL plasma cholesterol levels (r = 0.75; P = 0.001). The proportion of members of the family Coriobacteriaceae decreased when the hamsters were fed GSL and showed a high positive association with non-HDL plasma cholesterol levels (r = 0.84; P = 0.0002). These correlations were more significant than those between daily GSL intake and animal metabolic markers, implying that the dietary effects on host cholesterol metabolism are conferred, at least in part, through an effect on the gut microbiota. This study provides evidence that modulation of the gut microbiota-host metabolic interrelationship by dietary intervention has the potential to improve mammalian cholesterol homeostasis, which has relevance for cardiovascular health.

Proteogenomic landscape of squamous cell lung cancer.

How genomic and transcriptomic alterations affect the functional proteome in lung cancer is not fully understood. Here, we integrate DNA copy number, somatic mutations, RNA-sequencing, and expression proteomics in a cohort of 108 squamous cell lung cancer (SCC) patients. We identify three proteomic subtypes, two of which (Inflamed, Redox) comprise 87% of tumors. The Inflamed subtype is enriched with neutrophils, B-cells, and monocytes and expresses more PD-1. Redox tumours are enriched for oxidation-reduction and glutathione pathways and harbor more NFE2L2/KEAP1 alterations and copy gain in the 3q2 locus. Proteomic subtypes are not associated with patient survival. However, B-cell-rich tertiary lymph node structures, more common in Inflamed, are associated with better survival. We identify metabolic vulnerabilities (TP63, PSAT1, and TFRC) in Redox. Our work provides a powerful resource for lung SCC biology and suggests therapeutic opportunities based on redox metabolism and immune cell infiltrates.

Early transcriptional response of human ovarian and fallopian tube surface epithelial cells to norepinephrine.

Evidence from human and animal studies suggests that chronic behavioral stress and resulting activation of the sympathetic nervous system may influence initiation and progression of tumors. However, the underlying mechanisms for these observations are poorly understood. The purpose of this study is to explore the effects of adrenergic signaling on cell line models derived from normal cells presumed to originate epithelial ovarian cancers. Here we explored the effects of the stress-related hormone, norepinephrine, on the transcriptional program of normal immortalized ovarian (iOSE) and fallopian tube (iFTSEC) surface epithelial cells. Analysis of RNA-Seq data of treated and untreated cells revealed a significant overlap between the responses in iOSE and iFTSEC cells. Most genes modulated by norepinephrine in ovarian and fallopian tube epithelial cells are already expressed in normal ovarian and fallopian tissue and cells. For several genes, expression changes were reflected at the protein level. Genes in immune-related and developmental pathways were enriched in the set of genes modulated by norepinephrine. We identified HOXA5, SPIB, REL, SRF, SP1, NFKB1, MEF2A, E2F1, and EGR1 transcription factor binding sites to be highly enriched in our dataset. These data represent the early transcriptional response to norepinephrine in cells postulated to originate epithelial ovarian cancer.

Mutational heterogeneity in non-serous ovarian cancers.

Epithelial ovarian cancer is a leading cause of death in gynecological cancers. While several systematic studies have revealed the mutation landscape of serous epithelial ovarian cancer, other non-serous subtypes of the disease have not been explored as extensively. Here we conduct exome sequencing of nine non-serous epithelial ovarian tumors (six endometrioid and three mucinous) and their corresponding normal DNA as well as a tumor-only granulosa cell sample. We integrated the exome data with targeted gene sequencing for 1,321 genes selected for their involvement in cancer from additional 28 non-serous ovarian tumors and compared our results to TCGA ovarian serous cystadenocarcinoma and uterine corpus endometrial carcinomas. Prevalence of TP53 mutations in non-serous was much lower than in serous epithelial OC, whereas the prevalence of PIK3CA, PIK3R1, PTEN, CTNNB1, ARID1A, and KRAS was higher. We confirmed the high prevalence of FOXL2 and KRAS mutations in granulosa cell tumors and in mucinous tumors, respectively. We also identified POLE proofreading domain mutations in three endometrioid ovarian tumors. These results highlight mutational differences between serous and non-serous ovarian cancers, and further distinguish different non-serous subtypes.

Identification of a putative operon involved in fructooligosaccharide utilization by Lactobacillus paracasei.

The growth and activity of some Lactobacillus and Bifidobacterium strains are stimulated by the presence of nondigestible fructooligosaccharides (FOS), which are selectively fermented by specific intestinal bacteria. Consumption of FOS, therefore, enriches for those bacteria that possess metabolic pathways necessary for FOS metabolism. In this study, a DNA microarray consisting of 7,680 random genomic library fragments of Lactobacillus paracasei 1195 was used to examine genes involved in the utilization of FOS in this organism. Differential expression profiles between cells grown on FOS and those grown on glucose provided a basis for identifying genes specifically induced by FOS. Several of the FOS-induced genes shared sequence identity with genes encoding beta-fructosidases and components of phosphoenolpyruvate-dependent phosphotransferase systems (PTS). These genes were organized in a putative operon, designated the fos operon, that may play an essential role in FOS utilization. The complete 7,631-bp nucleotide sequence of the putative fos operon was determined and consists of fosABCDXE genes, which encode a putative fructose/mannose PTS (FosABCDX) and a beta-fructosidase precursor (FosE). The latter contains an N-terminal signal peptide sequence and cell wall sorting signals at the C-terminal region, suggesting its localization at the cell wall. Inactivation of the fosE gene led to impaired growth on FOS and other beta-fructose-linked carbohydrates. Transcriptional analysis by reverse transcriptase PCR suggested that fosABCDXE was cotranscribed as a single mRNA during growth on FOS. Expression array analysis revealed that when glucose was added to FOS-grown cells, transcription of the FOS-induced genes was repressed, indicating that FOS metabolism is subject to catabolite regulation.

Functional consequences of genome evolution in Listeria monocytogenes: the lmo0423 and lmo0422 genes encode sigmaC and LstR, a lineage II-specific heat shock system.

Listeria monocytogenes strains belonging to phylogenetic lineage II (serotypes 1/2a, 1/2c, and 3a) carry a lineage-specific genome segment encoding a putative sigma subunit of RNA polymerase (lmo0423, herein referred to as sigC), a gene of unknown function (lmo0422) similar to the padR family of regulators, and a gene that is similar to the rodA-ftsW family of cell wall morphology genes (lmo0421). To understand the function of this set of genes, their expression patterns and the effects of null mutations in the lineage II L. monocytogenes strain 10403S were examined. The data are consistent with the three genes comprising an operon (the sigC operon) that is highly induced by temperature upshift. The operon is transcribed from three different promoters, the proximal of which (P1) depends upon sigC itself. Null mutations in sigC or lmo0422 increase the death rate at lethal temperatures and cause loss of thermal adaptive response, whereas the lmo0421 mutation causes only a loss of the adaptive response component. Only the sigC mutation affects transcription from the P1 promoter, whereas ectopic expression of lmo0422 from the P(SPAC) promoter complements the individual lmo0422 and sigC null mutations, showing that lmo0422 is the actual thermal resistance regulator or effector while sigC provides a mechanism for temperature-dependent transcription of lmo0422 from P1. Our genetic and phylogenetic analyses are consistent with lmo0422-renamed lstR (for lineage-specific thermal regulator)-and sigC comprising a system of thermal resistance that was ancestral to the genus Listeria and was subsequently lost during divergence of the lineage I L. monocytogenes population.

Regulation of transcription of compatible solute transporters by the general stress sigma factor, sigmaB, in Listeria monocytogenes.

Listeria monocytogenes is well known for its durable physiological characteristics, which allow the organism to grow at low temperature and pH and high osmolarity. Growth under high osmolarity depends on the accumulation of compatible solutes, among which glycine betaine and carnitine are the preferred solutes for this organism. Three different transport systems, Gbu, BetL, and OpuC, have been identified in L. monocytogenes which serve to scavenge the preferred compatible solutes. The general stress response regulator sigma(B) has been shown to play an important role in osmotic adaptation in L. monocytogenes, presumably by directing transcription from one or more of the solute transport genes. In the studies presented here, we have used primer extension analyses to identify the promoter elements responsible for transcription of the opuC, gbuA, and betL genes. All three genes are osmotically inducible to some degree. betL is transcribed from a sigma(B)-independent promoter, while gbuA is transcribed from dual promoters, one of which is sigma(B) dependent. opuC is transcribed exclusively from a sigma(B)-dependent promoter. The betL promoter is similar in sequence to the sigma(B)-independent gbuAP1 promoter. Kinetic analysis of transcript accumulation after osmotic upshift demonstrated that sigma(B)-dependent transcripts from gbuAP2 and sigB accumulate for an extended period after upshift, suggesting that sigma(B) activity may provide a mechanism for sustained high-level expression during osmotic challenge. In contrast to osmotic upshift, expression from the sigma(B)-dependent opuC and gbuAP2 promoters after temperature upshift and ethanol stress was minimal, suggesting that additional mechanisms may also participate in regulating transcription from these sigma(B)-dependent promoters.

Suppressors of transcriptional transgenic silencing in Chlamydomonas are sensitive to DNA-damaging agents and reactivate transposable elements.

In the unicellular green alga Chlamydomonas reinhardtii, the epigenetic silencing of transgenes occurs, as in land plants, at both the transcriptional and posttranscriptional levels. In the case of single-copy transgenes, transcriptional silencing takes place without detectable cytosine methylation of the introduced DNA. We have isolated two mutant strains, Mut-9 and Mut-11, that reactivate expression of a transcriptionally silenced single-copy transgene. These suppressors are deficient in the repression of a DNA transposon and a retrotransposon-like element. In addition, the mutants show enhanced sensitivity to DNA-damaging agents, particularly radiomimetic chemicals inducing DNA double-strand breaks. All of these phenotypes are much more prominent in a double mutant strain. These observations suggest that multiple partly redundant epigenetic mechanisms are involved in the repression of transgenes and transposons in eukaryotes, presumably as components of a system that evolved to preserve genomic stability. Our results also raise the possibility of mechanistic connections between epigenetic transcriptional silencing and DNA double-strand break repair.