Reversible Epigenetic Mechanisms Research Articles

Abstract PO5-06-12: Genetic screen identifies epigenetic drivers of breast cancer brain metastasis

Abstract Metastatic breast cancer remains a major cause of cancer-related deaths in women, and there are few effective therapies against this advanced disease. Brain metastases, found in 15~35% of patients with breast cancer, are often incurable. Accumulating evidence suggests that key steps of tumor progression and metastasis are controlled by reversible epigenetic mechanisms. To systematically identify the druggable epigenetic regulators essential for breast cancer brain metastasis, we conducted an in vivo shRNA screen and identified multiple epigenetic regulators, including RNA acetyltransferase NAT10 as drivers of breast cancer brain metastasis. Knockdown of NAT10 significantly restrains breast cancer cell proliferation and migration in vitro and brain metastases in vivo. NAT10 promotes breast cancer cell growth through its RNA helicase activity but not its acetyltransferase activity. Integrative analyses of transcriptomics, proteomics, and NAT10 eCLIP-Seq data identify NAT10 downstream targets critical for breast cancer progression and metastasis. Thus, these results reveal novel therapeutic strategies to treat metastatic breast cancer. Citation Format: Jocelyn Chen, Wesley Cai, Peng Xu, Don Nguyen, Qin Yan. Genetic screen identifies epigenetic drivers of breast cancer brain metastasis [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-06-12.

Histone acylations as a mechanism for regulation of intestinal epithelial cells.

Histone post-translational modifications are reversible epigenetic mechanisms that regulate chromatin structure and gene transcription. In recent years, in addition to the well-characterized histone acetylation, new acylations such as propionylation, crotonylation, butyrylation and beta-hydroxybutyrylation have been described and explored in different cell types at contexts of health and disease. Understanding how histone acylations contribute to gene expression regulation is especially important in intestinal epithelial cells (IECs) because they receive many different signals from other cells and the external environment and must adapt to maintain essential functions such as nutrient and water absorption, maintenance of tolerance and protection against pathogens. In this review, we describe how cells regulate these modifications, how they are recognized by other proteins and impact gene expression. We summarize recent studies that explored the role of these distinct epigenetic marks in the regulation of IECs and discuss their biological importance for the intestinal epithelium's adaptations to changes in metabolism and to respond to environmental signals provided, for example, by the diet, components of the intestinal microbiota and pathogens. Finally, we discuss how the histone acylations are affected by inflammatory signals and how this knowledge may provide new targets for treatment of pathologies such as the inflammatory bowel diseases.

Abstract TP6: Identification of Circulating Epigenetic Biomarkers for Seizure in Brain Arteriovenous Malformation by Methylome Profiling

Background: Brain arteriovenous malformations (bAVMs) are an important cause of intracranial hemorrhage, seizure, or other neurological deficits. Clinical heterogeneity of bAVM suggests a role for genetic modifiers that may associate with seizure risk. DNA methylation is a reversible epigenetic mechanism that regulates gene expression in development and disease. We hypothesized that DNA methylation in epilepsy-related genes is associated with seizure at presentation in bAVM patients. Methods: We performed a literature review in PubMed to identify epilepsy-related genes with potential epigenetic regulation using the search terms: epigenetics, DNA methylation, seizure, epilepsy. We selected 19 unruptured bAVM cases (mean age 44.9 y) enrolled in the bAVM study at UCSF, including 10 presenting with seizure. We performed methylome sequencing of genomic DNA extracted from whole blood using bisulfite sequencing services at Psomagen with ~20X coverage. Differentially Methylated Regions (DMR) mapping to or nearby the candidate seizure genes were identified by comparing the average methylation ratio in bAVM cases presenting with and without seizure by t-test. DMRs with a delta average methylation ratio between the two groups ≥

Human WDR5 promotes breast cancer growth and metastasis via KMT2-independent translation regulation.

Metastatic breast cancer remains a major cause of cancer-related deaths in women, and there are few effective therapies against this advanced disease. Emerging evidence suggests that key steps of tumor progression and metastasis are controlled by reversible epigenetic mechanisms. Using an in vivo genetic screen, we identified WDR5 as an actionable epigenetic regulator that is required for metastatic progression in models of triple-negative breast cancer. We found that knockdown of WDR5 in breast cancer cells independently impaired their tumorigenic as well as metastatic capabilities. Mechanistically, WDR5 promotes cell growth by increasing ribosomal gene expression and translation efficiency in a KMT2-independent manner. Consistently, pharmacological inhibition or degradation of WDR5 impedes cellular translation rate and the clonogenic ability of breast cancer cells. Furthermore, a combination of WDR5 targeting with mTOR inhibitors leads to potent suppression of translation and proliferation of breast cancer cells. These results reveal novel therapeutic strategies to treat metastatic breast cancer.

The roles of epigenetics in cancer progression and metastasis.

Cancer metastasis remains a major clinical challenge for cancer treatment. It is therefore crucial to understand how cancer cells establish and maintain their metastatic traits. However, metastasis-specific genetic mutations have not been identified in most exome or genome sequencing studies. Emerging evidence suggests that key steps of metastasis are controlled by reversible epigenetic mechanisms, which can be targeted to prevent and treat the metastatic disease. A variety of epigenetic mechanisms were identified to regulate metastasis, including the well-studied DNA methylation and histone modifications. In the past few years, large scale chromatin structure alterations including reprogramming of the enhancers and chromatin accessibility to the transcription factors were shown to be potential driving force of cancer metastasis. To dissect the molecular mechanisms and functional output of these epigenetic changes, it is critical to use advanced techniques and alternative animal models for interdisciplinary and translational research on this topic. Here we summarize our current understanding of epigenetic aberrations in cancer progression and metastasis, and their implications in developing new effective metastasis-specific therapies.

Identification and Characterization of Epigenetic Regulators of Breast Cancer Metastasis to the Brain

Breast cancer is the second leading cause of cancer-related deaths in the US, primarily because breast cancer cells metastasize multiple vital organs, including the brain. Brain metastases, refractory to chemotherapy, occur in approximately 15-35% of metastatic breast cancer cases. Current clinical management of brain metastases is limited to radiation therapy and surgical resection, and the median survival time for patients is very short. Therefore, a better therapeutic strategy against the disease is urgently needed. Emerging evidence suggests that metastasis's key steps are controlled by reversible epigenetic mechanisms, which could be targeted to prevent and treat the condition. Using a systematic in vivo shRNA screening approach, which targets around 100 actionable epigenetic regulators, we have identified several potential drivers and suppressors of breast cancer brain metastasis. Interestingly, one of the candidate regulators, N-acetyltransferase 10 (NAT10), plays a role in catalyzing acetylation on specific RNA and protein substrates, while its regulatory mechanisms in breast cancer remain unclear. We found that knockdown of NAT10 suppresses breast cancer cell proliferation and migration in vitro and brain metastasis in vivo. We are leveraging transcriptomic and proteomic analyses to uncover key biological pathways regulated by NAT10. Here, we have demonstrated that this screening strategy is potent and can be applied to identify critical epigenetic regulators in other cancer types or metastatic organs. Our results allowed us to uncover the metastatic organotropism of the epigenes and develop a novel therapeutic regimen against metastatic diseases.

Identification of potential histone deacetylase1 (HDAC1) inhibitors using multistep virtual screening approach including SVM model, pharmacophore modeling, molecular docking and biological evaluation

Histone Deacetylases (HDACs) play a significant role in the regulation of gene expression by modifying histones and non-histone substrates. Since they are key regulators in the reversible epigenetic mechanism, they are considered as promising drug targets for the treatment of various cancers. In the present study, we have developed a workflow for identification of HDAC1 inhibitors using a multistage virtual screening approach from Maybridge and Chembridge chemical library. Initially, a support vector machine based classification model was generated, followed by generation of a zinc-binding group (ZBG) based pharmacophore model. The hits screened from these models were further subjected to molecular docking. Finally, a set of twenty-three molecules were selected from Maybridge and Chembridge library. The biological evaluation of these hits revealed that three out of the twenty-three tested compounds are showing HDAC1 inhibition along with the moderate anti-proliferative activity. It was found that the identified inhibitors are exerting chromosomal loss effect in growing yeast cells. Further, to extend the activity spectrum of the identified inhibitors, the optimization guidelines were drawn with the hydration site mapping approach by using in silico tool Watermap.Communicated by Ramaswamy H. Sarma

KLF11 is an Epigenetic Mediator of DRD2/Dopaminergic Signaling in Endometriosis.

Endometriosis is a heterogeneous, recalcitrant disease that affects 10% of reproductive-age women. Resistance to conventional therapy critically raises the need for novel treatment options that target specific, dysregulated underlying molecular mechanisms. Dopamine receptor 2 (DRD2) has been shown to be associated with vascularity and fibrosis in endometriosis. Transcription factor KLF11 has been implicated in the pathogenesis of several human endocrine and reproductive tract diseases including endometriosis. KLF11 recruits epigenetic cofactors for regulation of target genes; dysregulation of critical target genes and associated signaling pathways results in diverse disease phenotypes. KLF11 regulates the expression of DRD2 in neurons. We investigated the regulation of DRD2 by KLF11 in the established eutopic and ectopic endometrial cell lines as well as in an animal model of endometriosis. KLF11 binding and activation of the DRD2 promoter was conserved across species. Promoter activation was reflected in correspondingly increased gene expression in an endometrial cell line and in primary endometriotic cells. In vivo, disease relevance was further evaluated in a surgically induced murine endometriotic model using Klf11-/- and wild-type mice. Consistent with loss of Klf11-mediated activation, lesions in Klf11-/- animals were associated with progressive fibrosis and decreased Drd2 expression. KLF11 binds specific epigenetic corepressors to repress several target genes. Activation of DRD2 by KLF11 could not be explained simply by loss of corepressor binding and is thus likely due to selective coactivator recruitment; identification of the precise pathway is the focus of ongoing investigation. Characterization of pharmacologically reversible epigenetic regulatory mechanisms has translational relevance in health and disease.

The Combined Bisulfite Restriction Analysis (COBRA) Assay for the Analysis of Locus-Specific Changes in Methylation Patterns.

DNA methylation is a heritable but reversible epigenetic mechanism of control over gene expression. The level of DNA methylation of specific genomic regions correlates with chromatin condensation, the level of gene expression, and in some cases genome stability and the frequency of homologous recombination. Here, we describe the combined bisulfite restriction analysis (COBRA) assay that allows analyzing the methylation status at a specific locus. The protocol consists of the following major steps: bisulfite conversion of non-methylated cytosines to uracils, the locus-specific PCR amplification of converted DNA, restriction digestion, the analysis of restriction patterns on the gel, and the quantification of these restriction patterns using ImageJ or a similar program.

IMPS-13IMMUNOLOGIC PROFILING OF PEDIATRIC CNS MALIGNANCIES FOR THE RATIONAL SELECTION OF IMUNOTHERAPEUTIC TRIALS

BACKGROUND: Although clinical trials have resulted in significant improvements in childhood brain cancer treatments, further progress using strategically targeted, potentially less toxic modalities are needed. Immunotherapy is one such emerging treatment approach. The degree of tumor-infiltrating cytotoxic T-lymphocytes (CTLs) directly correlates with prognosis for several cancer types. MHC Class I is down-regulated in 40-90% of adult cancers, rendering them resistant to CTL attack, correlating with poorer prognosis. MHC I expression in medulloblastomas is associated with poor prognostic factors, a pattern opposite of most other cancer-MHC I associations described. Natural Killer (NK)-cell based therapies, dependent upon low MHC I expression, are therefore proposed as an immunotherapeutic approach to medulloblastomas. Very little literature exists classifying MHC I expression in other pediatric brain cancers, despite several ongoing trials using MHC I-dependent strategies. This lack of data raises uncertainty of which immunotherapies are optimal for these diseases. Toward this end, we classified key immunologic characteristics of pediatric brain tumors. METHODS: We interrogated pre-clinical and clinical mRNA microarray databases to identify tumor types which exhibit MHC I and the associated b-2-microglobulin molecule expression. We up-regulated MHC I expression in vitro in pediatric CNS tumor cell lines and identified intra-tumoral innate and adaptive immunologic cellular infiltrates within pediatric CNS tumor samples through immunohistochemistry. RESULTS: We found that atypical teratoid/rhabdoid tumors (AT/RT) and ependymomas exhibit decreased, whereas medulloblastomas and glioblastomas express higher MHC I mRNA levels than most other pediatric cancer types. We discovered MHC I can be up-regulated in vitro in medulloblastomas and AT/RT's, indicating a reversible epigenetic mechanism for down-regulation. There were decreased NK and regulatory T-cell, while increased CTL infiltrates in recurred versus diagnostic pediatric glioblastoma tumor samples. CONCLUSIONS: The immunologic profiling of pediatric brain cancers will guide rational selection of immune-based therapies, serving as an important biomarker for patient inclusion in immunotherapeutic trials.

Abstract A43: Immunologic profiling of pediatric malignancies for plausible immunotherapeutic trial selection

Abstract Although clinical trials have resulted in significant improvement in the treatment of childhood cancers, further progress using more targeted and less toxic modalities requires a better understanding of the biology of these diseases, including how they develop the ability to overcome rejection by the immune system. In adult cancers, immunotherapies are gaining traction with increasing reports of antitumor efficacy. Cytotoxic T lymphocytes (CTLs) have a major role in the immune response to tumors and require antigenic presentation in the context of Major Histocompatibility Complex (MHC) Class I. Studies have reported the degree of these tumor-infiltrating lymphocytes to be directly correlated with prognosis. Prior studies have also shown that MHC Class I is down-regulated in 40-90% of adult cancer cases, making them resistant to an immune attack by CTLs, and have indicated that MHC Class I expression down-regulation is associated with poorer prognosis. Some immunotherapeutic approaches, such as chimeric antigen receptors and bi-specific antibodies, are MHC Class I-independent. Others, such as vaccines and the adoptive transfer of tumor-specific T-cells, depend upon antigenic peptide presentation by MHC Class I to be effective. In addition to direct oncolysis, oncolytic viruses exert their therapeutic effects in part by inducing a T-cell response against tumor-associated antigens, rendering their benefits to be also somewhat MHC-dependent. Oncolytic herpes simplex virus (oHSV) therapy is an emerging immunotherapeutic platform in clinical trials, and our lab has previously characterized implantable mouse RMS tumor cell lines for their suitability as immunocompetent models for the study of the adaptive immune response to oHSV therapy. For the appropriate enrollment of patients onto immunotherapeutic clinical trials, it is imperative to know the MHC Class I expression on tumor cells, and, for virotherapy, the effects of infection on its expression. We sought to characterize MHC Class I expression in pediatric malignancies and queried mRNA microarray databases including clinical and preclinical datasets. We characterized the immunologic profile of pediatric malignancies through the identification of tumor types which exhibit MHC Class I and the associated β-2-microglobulin molecule expression. Preliminary data suggest that sarcomas, including Ewing sarcoma, osteosarcoma, and both embryonal and alveolar rhabdomyosarcomas (RMS), Wilms' tumors, and some brain tumors, such as atypical teratoid/ rhabdoid tumors (AT/RT) and ependymomas, exhibit decreased MHC Class I mRNA levels relative to other cancer types. We discovered that MHC Class I can be up-regulated in vitro in many human pediatric cancer cell lines, indicating a reversible epigenetic mechanism for down-regulation. Our immunocompetent murine RMS models suggest that the oHSV in vivo therapeutic effect is largely T-cell dependent. The most resistant model exhibited a cellular infiltrate dominated by CD4+ T cells, while the sensitive model demonstrated higher baseline CD8+ T cells. In fact, the best therapeutic response, including complete tumor rejection and resistance to tumor re-challenge, correlated with highest CTL infiltrate and the highest MHC Class I expression. These data support the likelihood that the oHSV therapeutic effect is not necessarily governed by susceptibility or permissiveness to oHSV oncolysis, but rather by the effect of oHSV on tumor cell-microenvironmental immunologic interactions. We hypothesize that MHC Class I status may serve as an important biomarker for patient inclusion in therapeutic trials of MHC-dependent immunotherapies such as oncolytic viral therapy, and modulation of MHC Class I expression will be a promising strategy to enhance immunotherapeutic efficacy. Citation Format: Kellie B. Haworth, Jennifer L. Leddon, Chun-Yu Chen, Timothy P. Cripe. Immunologic profiling of pediatric malignancies for plausible immunotherapeutic trial selection. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A43.

The impact of Polycomb group (PcG) and Trithorax group (TrxG) epigenetic factors in plant plasticity.

Current advances indicate that epigenetic mechanisms play important roles in the regulatory networks involved in plant developmental responses to environmental conditions. Hence, understanding the role of such components becomes crucial to understanding the mechanisms underlying the plasticity and variability of plant traits, and thus the ecology and evolution of plant development. We now know that important components of phenotypic variation may result from heritable and reversible epigenetic mechanisms without genetic alterations. The epigenetic factors Polycomb group (PcG) and Trithorax group (TrxG) are involved in developmental processes that respond to environmental signals, playing important roles in plant plasticity. In this review, we discuss current knowledge of TrxG and PcG functions in different developmental processes in response to internal and environmental cues and we also integrate the emerging evidence concerning their function in plant plasticity. Many such plastic responses rely on meristematic cell behavior, including stem cell niche maintenance, cellular reprogramming, flowering and dormancy as well as stress memory. This information will help to determine how to integrate the role of epigenetic regulation into models of gene regulatory networks, which have mostly included transcriptional interactions underlying various aspects of plant development and its plastic response to environmental conditions.

Abstract 3518: Frequent mutation and deletion of GATA4 in hepatocellular carcinoma represses proliferation terminating late-maturation genes CEBPD and HNF4A by reversible epigenetic mechanisms.

Abstract One reason that hepatocellular carcinoma (HCC) is resistant to therapy is frequent mutation or deletion of master regulators of apoptosis (e.g., TP53) that mediate the actions of multiple cancer drugs. Maturation genes are another family of genes which mediate cell cycle exit. If unlike key apoptosis genes these proliferation-terminating genes are genetically intact but epigenetically repressed in HCC, then understanding and reversing the mechanisms of aberrant repression could form the basis for novel treatment. Hence, with integrated microarray analyses and Sanger sequencing of 46 primary HCC samples (25 HepB, 16 non-viral) and adjacent non-malignant liver controls, complemented by in vitro studies and confirmatory analyses of public databases, this study sought to identify pathways by which hepatocyte late-maturation MYC-antagonist genes are suppressed in HCC, and to examine the translational implications. The hepatocyte late-maturation genes CEBPD and HNF4A are known to antagonize MYC and terminate proliferation. CEBPD and HNF4A expression were significantly decreased in HCC compared to adjacent normal liver (p<0.0001) even though the CEBPD and HNF4A loci were not deleted in any cases. Consistent with epigenetic repression, there was significant hypermethylation of CpG in the promoters for these genes. Two master drivers of hepatocyte commitment that regulate these maturation genes are FOXA2 and GATA4. FOXA2, and also FOXA1/3, were expressed at higher levels in HCC than in adjacent normal liver. However, GATA4 expression was significantly decreased. In 39 HCC cases with karyotype analyzed by SNP array, there were no instances of FOXA2 deletion. In contrast, GATA4 was deleted in 14/39 cases (36%) and mutated (non-synonymous missense or frameshift mutations in exons 4 or 6, e.g., 11614516G>A:S357T) in 17/46 cases (37%) (total GATA4 genetic abnormality 25/43 [58%]). Although GATA4 was not mutated/deleted in every case, all 46 HCC demonstrated decreased expression of HNF4A and highly conserved HNF4A target genes, indicating that this pathway is targeted in most if not all HepB pos or neg HCC. Extrinsic expression of GATA4 in HCC cells (HepG2, PLC) renewed HNF4A and CEBPD expression, decreased MYC and SKP2, increased p27/CDKN1B, and induced cell cycle exit. GATA4 may cooperate with FOXA2 to regulate corepressor/coactivator exchange at target genes. Accordingly, disrupting corepressor function with non-cytotoxic, DNMT1 depleting concentrations of decitabine (0.2-0.5 microM) compensated for GATA4 deficiency and activated CEBPD, HNF4A, decreased MYC, increased p27/CDKN1B, and induced cell cycle exit. GATA4 mutation and deletion in HCC represses HNF4A and CEBPD by therapeutically reversible mechanisms, offering a p53-independent alternative to ineffective apoptosis-based therapy. Citation Format: Francis O. Enane, Marissa Teo, Hideki Makishima, Zhenbo Hu, Han Chong Toh, Yogen Saunthararajah, Joanna Ng, Chit Lai, Soo Fan Ang, Lim Kiat Hon. Frequent mutation and deletion of GATA4 in hepatocellular carcinoma represses proliferation terminating late-maturation genes CEBPD and HNF4A by reversible epigenetic mechanisms. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3518. doi:10.1158/1538-7445.AM2013-3518

Epigenetic Remedies by Dietary Phytochemicals Against Inflammatory Skin Disorders: Myth or Reality?

While many botanicals have been used during thousands of years in various cultures for the treatment of several inflammatory conditions, wound healing or preserving skin beauty, their active ingredients and their mechanisms of action are less well characterized. It is known that throughout life, environmental conditions and dietary compounds influence gene expression. Only recently it has been observed that exposure to specific phytochemicals can affect gene expression via reversible epigenetic mechanisms and gets recorded in our "epigenome" through life. Epigenetics refers to heritable phenotypical differences or changes in gene expression that are not attributable to changes in DNA sequence, but rather depend on variations in DNA methylation, chromatin structure or microRNA profiles. As such, our dietary epigenetic imprint superposed on our genome may rewire gene expression patterns in the body and the host immune system, and protect against inflammatory disorders, cancer and ageing. This has recently launched reexploration of nutritional, botanical or phytopharmaceutical compounds for epigenetic effects to identify promising nutraceuticals or cosmeceuticals which could (re)program stem cell differentiation, wound healing, skin regeneration, tissue homeostasis, or "correct" epigenetic marks responsible for inflammatory skin disorders and ageing.

DNA methyltransferase-mediated transcriptional silencing in malignant glioma: a combined whole-genome microarray and promoter array analysis

Epigenetic inactivation of tumor suppressor genes is a common feature in human cancer. Promoter hypermethylation and histone deacetylation are reversible epigenetic mechanisms associated with transcriptional regulation. DNA methyltransferases (DNMT1 and DNMT3b) regulate and maintain promoter methylation and are overexpressed in human cancer. We performed whole-genome microarray analysis to identify genes with altered expression after RNAi-induced suppression of DNMT in a glioblastoma multiforme (GBM) cell line. We then identified genes with both decreased expression and evidence of promoter CpG island hypermethylation in GBM tissue samples using a combined whole-genome microarray transcriptome analysis in conjunction with a promoter array analysis after DNA immunoprecipitation with anti-5-methylcytidine. DNMT1 and 3b knockdown resulted in the restored expression of 308 genes that also contained promoter region hypermethylation. Of these, 43 were also found to be downregulated in GBM tissue samples. Three downregulated genes with hypermethylated promoters and restored expression in response to acute DNMT suppression were assayed for methylation changes using bisulfite sequence analysis of the promoter region after chronic DNMT suppression. Restoration of gene expression was not associated with changes in promoter region methylation, but rather with changes in histone methylation and chromatin conformation. Two of the identified genes exhibited growth suppressive activity in in vitro assays. Combining targeted genetic manipulations with comprehensive genomic and expression analyses provides a potentially powerful new approach for identifying epigenetically regulated genes in GBM.

Cryopreservation induces temporal DNA methylation epigenetic changes and differential transcriptional activity in Ribes germplasm

The physiological and molecular mechanisms associated with acclimation and survival have been examined in four Ribes genotypes displaying differential cryotolerance. Changes in DNA methylation, nucleic acid and nucleoside composition were determined during acclimation and recovery of in vitro shoot-meristems from cryopreservation. DNA methylation was induced in the tolerant genotype, while demethylation was evident in sensitive genotypes. This response initially occurred during sucrose simulated acclimation, with progressive changes as shoots recovered from successive stages of the encapsulation-dehydration protocol. These methylation patterns existed in the initial vegetative cycle but regressed to control values following subculture, indicating the changes in DNA methylation to be a reversible epigenetic mechanism. RNA levels indicating transcriptional activity during the acclimation of nodal tissue are inversely linked to methylation changes, where activity appears to be up-regulated in the cryosensitive genotypes. Conversely, cryopreserved shoots show increased levels of both RNA and DNA methylation in the cryotolerant genotypes. Other nucleosides show post-transcriptional activity corresponds with tolerance during acclimation and cryopreservation. These observations connect physiological attributes to differential molecular changes in Ribes, the implications of which are discussed in relation to cryopreservation-induced apoptosis and genetic stability.

Potent Selection of Antigen Loss Variants of B16 Melanoma following Inflammatory Killing of MelanocytesIn vivo

We have reported that i.d. injection of plasmids encoding hsp70 and a suicide gene transcriptionally targeted to melanocytes generates specific proinflammatory killing of melanocytes. The resulting CD8+ T cell response eradicates systemically established B16 tumors. Here, we studied the consequences of that CD8+ T cell response on the phenotype of preexisting tumor. In suboptimal protocols, the T cell response selected B16 variants, which grow extremely aggressively, are amelanotic and have lost expression of the tyrosinase and tyrosinase-related protein 2 (TRP-2) antigens. However, expression of other melanoma-associated antigens, such as gp100, was not affected. Antigen loss could be reversed by long-term growth in culture away from immune-selective pressures or within 96 hours by treatment with the demethylating agent 5-azacytidine (5-Aza). When transplanted back into syngeneic animals, variants were very poorly controlled by further vaccination. However, a combination of vaccination with 5-Aza to reactivate antigen expression in tumors in situ generated highly significant improvements in therapy over treatment with vaccine or 5-Aza alone. These data show that inflammatory killing of normal cells activates a potent T cell response targeted against a specific subset of self-antigens but can also lead to the immunoselection of tumor variants. Moreover, our data indicate that emergence of antigen loss variants may often be due to reversible epigenetic mechanisms within the tumor cells. Therefore, combination therapy using vaccination and systemic treatment with 5-Aza or other demethylating agents may have significant therapeutic benefits for antitumor immunotherapy.

Epigenetic silencing of a foreign gene in nuclear transformants of Chlamydomonas.

The unstable expression of introduced genes poses a serious problem for the application of transgenic technology in plants. In transformants of the unicellular green alga Chlamydomonas reinhardtii, expression of a eubacterial aadA gene, conferring spectinomycin resistance, is transcriptionally suppressed by a reversible epigenetic mechanism(s). Variations in the size and frequency of colonies surviving on different concentrations of spectinomycin as well as the levels of transcriptional activity of the introduced transgene(s) suggest the existence of intermediate expression states in genetically identical cells. Gene silencing does not correlate with methylation of the integrated DNA and does not involve large alterations in its chromatin structure, as revealed by digestion with restriction endonucleases and DNase I. Transgene repression is enhanced by lower temperatures, similar to position effect variegation in Drosophila. By analogy to epigenetic phenomena in several eukaryotes, our results suggest a possible role for (hetero)chromatic chromosomal domains in transcriptional inactivation.

Factors affecting long-term stability of Moloney murine leukemia virus-based vectors

We have examined the long-term functional and structural stability of retroviral vectors in infected murine cells. We have used Moloney murine leukemia virus-based vectors epxressing human HPRT, firefly luciferase ( luc), and Escherichia coli β-galactosidase ( lacZ) as reporter genes, and the human HPRT and the transposon Tn5 neomycin resistance ( neo) gene as selectable markers. All vectors, whether single or double gene, yielded both stable and unstable clones. Stability of the proviruses was dependent on a number of factors, including the nature of the infected cell, the reporter gene, the integration site of the provirus, the relative positions of the component genes in multigene vectors, and the presence or absence of selection pressure. Selection pressure was helpful, but not universally effective, in maintaining provirus structural and functional integrity. Reporter gene expression from an internal promoter was likely to be unstable with or without selection for an upstream, LTR-driven neo gene. In some clones, loss of proviral gene expression was accompanied by deletions, while other inactive clones retained an apparently intact provirus. In the latter clones, treatment with 5-azacytidine failed to reactivate the reporter genes, but superinfection with helper virus resulted in the reappearance of transmissible vector, indicating a reversible epigenetic mechanism for proviral shutdown. The design of effective retroviral vectors and their possible use in vivo will require further characterization of these determinants of provirus stability.