Chromatin Alterations Research Articles

Comprehensive Analysis and Verification of the Prognostic Significance of Cuproptosis-Related Genes in Colon Adenocarcinoma.

Colon adenocarcinoma (COAD) is a frequently occurring and lethal cancer. Cuproptosis is an emerging type of cell death, and the underlying pathways involved in this process in COAD remain poorly understood. Transcriptomic and clinical data for COAD patients were collected from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. We investigated alterations in DNA and chromatin of cuproptosis-related genes (CRGs) in COAD. In order to identify predictive differentially expressed genes (DEGs) and various molecular subtypes, we used consensus cluster analysis. Through univariate, multivariate, and Lasso Cox regression analyses, four CRGs were identified. A risk prognostic model for cuproptosis characteristics was constructed based on four CRGs. This study also examined the association between the risk score and the tumor microenvironment (TME), the immune landscape, and drug sensitivity. We distinguished two unique molecular subtypes using consensus clustering analysis. We discovered that the clinical characteristics, prognosis, and TME cell infiltration characteristics of patients with multilayer CRG subtypes were all connected. The internal and external evaluations of the predicted accuracy of the prognostic model built using data derived from a cuproptosis risk score were completed at the same time. A nomogram and a clinical pathological analysis make it more useful in the field of medicine. A significant rise in immunosuppressive cells was observed in the high cuproptosis risk score group, with a correlation identified between the cuproptosis risk score and immune cell infiltration. Despite generally poor prognoses, the patients with a high cuproptosis risk but low tumor mutation burden (TMB), cancer stem cell (CSC) index, or microsatellite instability (MSI) may still benefit from immunotherapy. Furthermore, the cuproptosis risk score positively correlated with immune checkpoint gene expression. Analyzing the potential sensitivity to medications could aid in the development of clinical chemotherapy regimens and decision-making. CRGs are the subject of our in-depth study, which exposed an array of regulatory mechanisms impacting TME. In addition, we performed additional data mining into clinical features, prognosis effectiveness, and possible treatment medications. COAD's molecular pathways will be better understood, leading to more precise treatment options.

Skin senescence-from basic research to clinical practice.

The most recognizable implications of tissue aging manifest themselves on the skin. Skin laxity, roughness, pigmentation disorders, age spots, wrinkles, telangiectasia or hair graying are symptoms of physiological aging. Development of the senescent phenotype depends on the interaction between aging cells and remodeling of the skin's extracellular matrix (ECM) that contains collagen and elastic fiber. Aging changes occur due to the combination of both endogenous (gene mutation, cellular metabolism or hormonal agents) and exogenous factors (ultraviolet light, environmental pollutants, and unsuitable diet). However, overproduction of mitochondrial reactive oxygen species (ROS) is a key factor driving cellular senescence. Aging theories have disclosed a range of diverse molecular mechanisms that are associated with cellular senescence of the body. Theories best supported by evidence include protein glycation, oxidative stress, telomere shortening, cell cycle arrest, and a limited number of cell divisions. Accumulation of the ECM damage is suggested to be a key factor in skin aging. Every cell indicates a functional and morphological change that may be used as a biomarker of senescence. Senescence-associated β-galactosidase (SA-β-gal), cell cycle inhibitors (p16INK4a, p21CIP1, p27, p53), DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS), senescence-associated heterochromatin foci (SAHF), shortening of telomeres or downregulation of lamina B1 constitute just an example of aging biomarkers known so far. Aging may also be assessed non-invasively through measuring the skin fluorescence of advanced glycation end-products (AGEs). This review summarizes the recent knowledge on the pathogenesis and clinical conditions of skin aging as well as biomarkers of skin senescence.

Single-cell transcriptomes and chromatin accessibility of endothelial cells unravel transcription factors associated with dysregulated angiogenesis in systemic sclerosis

ObjectivesVasculopathy emerges early in systemic sclerosis (SSc) and links to endothelial cell (EC) injury and angiogenesis. Understanding EC transcriptomes and epigenomes is crucial for unravelling the mechanisms involved.MethodsTranscriptomes and chromatin...

The role of hexokinases in epigenetic regulation: altered hexokinase expression and chromatin stability in yeast

BackgroundHuman hexokinase 2 (HK2) plays an important role in regulating Warburg effect, which metabolizes glucose to lactate acid even in the presence of ample oxygen and provides intermediate metabolites to support cancer cell proliferation and tumor growth. HK2 overexpression has been observed in various types of cancers and targeting HK2-driven Warburg effect has been suggested as a potential cancer therapeutic strategy. Given that epigenetic enzymes utilize metabolic intermediates as substrates or co-factors to carry out post-translational modification of histones and nucleic acids modifications in cells, we hypothesized that altering HK2 expression could impact the epigenome and, consequently, chromatin stability in yeast. To test this hypothesis, we established genetic models with different yeast hexokinase 2 (HXK2) expression in Saccharomyces cerevisiae yeast cells and investigated the effect of HXK2-dependent metabolism on parental nucleosome transfer, a key DNA replication–coupled epigenetic inheritance process, and chromatin stability.ResultsBy comparing the growth of mutant yeast cells carrying single deletion of hxk1Δ, hxk2Δ, or double-loss of hxk1Δ hxk2Δ to wild-type cells, we firstly confirmed that HXK2 is the dominant HXK in yeast cell growth. Surprisingly, manipulating HXK2 expression in yeast, whether through overexpression or deletion, had only a marginal impact on parental nucleosome assembly, but a noticeable trend with decrease chromatin instability. However, targeting yeast cells with 2-deoxy-D-glucose (2-DG), a clinical glycolysis inhibitor that has been proposed as an anti-cancer treatment, significantly increased chromatin instability.ConclusionOur findings suggest that in yeast cells lacking HXK2, alternative HXKs such as HXK1 or glucokinase 1 (GLK1) play a role in supporting glycolysis at a level that adequately maintains epigenomic stability. While our study demonstrated an increase in epigenetic instability with 2-DG treatment, the observed effect seemed to occur dependent on non-glycolytic function of Hxk2. Thus, additional research is needed to identify the molecular mechanism through which 2-DG influences chromatin stability.

Machine learning approaches to detect hepatocyte chromatin alterations from iron oxide nanoparticle exposure

This study focuses on developing machine learning models to detect subtle alterations in hepatocyte chromatin organization due to Iron (II, III) oxide nanoparticle exposure, hypothesizing that exposure will significantly alter chromatin texture. A total of 2000 hepatocyte nuclear regions of interest (ROIs) from mouse liver tissue were analyzed, and for each ROI, 5 different parameters were calculated: Long Run Emphasis, Short Run Emphasis, Run Length Nonuniformity, and 2 wavelet coefficient energies obtained after the discrete wavelet transform. These parameters served as input for supervised machine learning models, specifically random forest and gradient boosting classifiers. The models demonstrated relatively robust performance in distinguishing hepatocyte chromatin structures belonging to the group exposed to IONPs from the controls. The study's findings suggest that iron oxide nanoparticles induce substantial changes in hepatocyte chromatin distribution and underscore the potential of AI techniques in advancing hepatocyte evaluation in physiological and pathological conditions.

Canine hyper-sociability structural variants associated with altered three-dimensional chromatin state

Strong selection on complex traits can lead to skewed trait means and reduced trait variability in populations. An example of this phenomenon can be evidenced in allele frequency changes and skewed trait distributions driven by persistent human-directed selective pressures in domesticated species. Dog domestication is linked to several genomic variants; however, the functional impacts of these variants may not always be straightforward when found in non-coding regions of the genome. Four polymorphic transposable elements (TE) found within non-coding sites along a 5 Mb region on canine CFA6 have evolved due to directional selection associated with heightened human-directed hyper-sociability in domesticated dogs. We found that the polymorphic TE in intron 17 of the canine GTF2I gene, which was previously reported to be negatively correlated with canid human-directed hyper-sociability, is associated with altered chromatin looping and hence distinct cis-regulatory landscapes. We reported supporting evidence of an E2F1-DNA binding peak concordant with the altered loop and higher expression of GTF2I exon 18, indicative of alternative splicing. Globally, we discovered differences in pathways regulating the extra-cellular matrix with respect to TE copy number. Overall, we reported evidence suggesting an intriguing molecular convergence between the emergence of hypersocial behaviors in dogs and the same genes that, when hemizygous, produce human Williams Beuren Syndrome characterized by cranio-facial defects and heightened social behaviors. Our results additionally emphasize the often-overlooked potential role of chromatin architecture in social evolution.

Large-scale discovery of chromatin dysregulation induced by oncofusions and other protein-coding variants.

Population-scale databases have expanded to millions of protein-coding variants, yet insight into their mechanistic consequences has lagged. Here we present PROD-ATAC, a high-throughput method for discovering the effects of protein-coding variants on chromatin regulation. A pooled variant library is expressed in a disease-agnostic cell line, and single-cell assay for transposase-accessible chromatin resolves each variant's effect on the chromatin landscape. Using PROD-ATAC, we characterized the effects of more than 100 oncofusions (cancer-causing chimeric proteins) and controls and revealed that chromatin remodeling is common to fusions spanning an enormous range of fusion frequencies. Furthermore, fusion-induced dysregulation can be context agnostic, as observed mechanisms often overlapped with cancer and cell-type-specific prior knowledge. We also showed that gain-of-function activity is common among oncofusions. This work begins to outline a global map of fusion-induced chromatin alterations. We suggest that there might be convergent mechanisms among disparate oncofusions and shared modes of dysregulation among fusions present in tumors at different frequencies. PROD-ATAC is generalizable to any set of protein-coding variants.

Comparison between open and closed systems for vitrification of individual sperm: assessing morphometric measurements and chromatin integrity

BackgroundClassic vitrification methods are not appropriate when there are minimal numbers of viable sperm, and the new methods emphasize the low semen volumes in these cases. The aim was to assess the efficacy of the cryotech as a device for freezing low sperm volume, through the two methods of open (OVS) and closed (CVS) vitrification systems.MethodsTesticular biopsy samples from 30 men with obstructive azoospermia (OA) were assigned to three groups fresh control (FC), OVS, and CVS. Testicular sperms were selected using an ICSI injection pipette and vitrified on the cryotech straws, containing one droplet of freezing medium. After warming, sperm head morphometric characterizations were evaluated with the MSOME technique. Sperm motility, membrane integrity, chromatin quality assessment including DNA fragmentation, Chromomycine A3 staining (CMA3), and Aniline Blue (AB) were assessed. Fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) was done to examine sperm acrosome integrity.ResultsThe mean sperm motility, viability, and sperm with intact acrosome reduced after vitrification, in both methods of CVS, and OVS, but the results were more promising in the closed method (p < 0.05). However, the variations were not significant between the two methods of cryopreservation, the OVS undergoes significant head dimensions changes compared to the control group (p < 0.05). The results also showed higher membrane, and chromatin abnormality after OVS (p < 0.05).ConclusionsThe overall post-thaw recovery of human testicular sperm proposes that CVS is more efficient for single sperm cryopreservation, while higher sperm viability, and lower alterations in chromatin, acrosome, and sperm head morphometry were seen compared to OVS.

Type II innate lymphoid cell plasticity contributes to impaired reconstitution after allogeneic hematopoietic stem cell transplantation

Type II innate lymphoid cells (ILC2s) maintain homeostasis and barrier integrity in mucosal tissues. In both mice and humans, ILC2s poorly reconstitute after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Determining the mechanisms involved in their impaired reconstitution could improve transplant outcomes. By integrating single-cell chromatin and transcriptomic analyses of transplanted ILC2s, we identify a previously unreported population of converted ILC1-like cells in the mouse small intestine post-transplant. Exposure of ILC2s to proinflammatory cytokines resulted in a mixed ILC1-ILC2 phenotype but was able to convert only a small population of ILC2s to ILC1s, which were found post-transplant. Whereas ILC2s protected against acute graft-versus-host disease (aGVHD) mediated mortality, infusion of proinflammatory cytokine-exposed ILC2s accelerated aGvHD. Interestingly, murine ILC2 reconstitution post-HSCT is decreased in the presence of alloreactive T cells. Finally, peripheral blood cells from human patients with aGvHD have an altered ILC2-associated chromatin landscape compared to transplanted controls. These data demonstrate that following transplantation ILC2s convert to a pro-pathogenic population with an ILC1-like chromatin state and provide insights into the contribution of ILC plasticity to the impaired reconstitution of ILC2 cells, which is one of several potential mechanisms for the poor reconstitution of these important cells after allo-HSCT.

Loss of Kmt2c or Kmt2d drives brain metastasis via KDM6A-dependent upregulation of MMP3

KMT2C and KMT2D, encoding histone H3 lysine 4 methyltransferases, are among the most commonly mutated genes in triple-negative breast cancer (TNBC). However, how these mutations may shape epigenomic and transcriptomic landscapes to promote tumorigenesis is largely unknown. Here we describe that deletion of Kmt2c or Kmt2d in non-metastatic murine models of TNBC drives metastasis, especially to the brain. Global chromatin profiling and chromatin immunoprecipitation followed by sequencing revealed altered H3K4me1, H3K27ac and H3K27me3 chromatin marks in knockout cells and demonstrated enhanced binding of the H3K27me3 lysine demethylase KDM6A, which significantly correlated with gene expression. We identified Mmp3 as being commonly upregulated via epigenetic mechanisms in both knockout models. Consistent with these findings, samples from patients with KMT2C-mutant TNBC have higher MMP3 levels. Downregulation or pharmacological inhibition of KDM6A diminished Mmp3 upregulation induced by the loss of histone–lysine N-methyltransferase 2 (KMT2) and prevented brain metastasis similar to direct downregulation of Mmp3. Taken together, we identified the KDM6A–matrix metalloproteinase 3 axis as a key mediator of KMT2C/D loss-driven metastasis in TNBC.

3D Chromatin Alteration by Disrupting β-Catenin/CBP Interaction Is Enriched with Insulin Signaling in Pancreatic Cancer.

The therapeutic potential of targeting the β-catenin/CBP interaction has been demonstrated in a variety of preclinical tumor models with a small molecule inhibitor, ICG-001, characterized as a β-catenin/CBP antagonist. Despite the high binding specificity of ICG-001 for the N-terminus of CBP, this β-catenin/CBP antagonist exhibits pleiotropic effects. Our recent studies found global changes in three-dimensional (3D) chromatin architecture in response to disruption of the β-catenin/CBP interaction in pancreatic cancer cells. However, an understanding of how the functional crosstalk between the antagonist and the β-catenin/CBP interaction affects changes in 3D chromatin architecture and, thereby, gene expression and downstream effects remains to be elucidated. Here, we perform Hi-C analyses on canonical and patient-derived pancreatic cancer cells before and after treatment with ICG-001. In addition to global alteration of 3D chromatin domains, we unexpectedly identify insulin signaling genes enriched in the altered chromatin domains. We further demonstrate that the chromatin loops associated with insulin signaling genes are significantly weakened after ICG-001 treatment. We finally elicit the deletion of a looping of IRS1-a key insulin signaling gene-significantly impeding pancreatic cancer cell growth, indicating that looping-mediated insulin signaling might act as an oncogenic pathway to promote pancreatic cancer progression. Our work shows that targeting aberrant insulin chromatin looping in pancreatic cancer might provide a therapeutic benefit.

New facets in the chromatin-based regulation of genome maintenance

The maintenance of genome integrity by DNA damage response machineries is key to protect cells against pathological development. In cell nuclei, these genome maintenance machineries operate in the context of chromatin, where the DNA wraps around histone proteins. Here, we review recent findings illustrating how the chromatin substrate modulates genome maintenance mechanisms, focusing on the regulatory role of histone variants and post-translational modifications. In particular, we discuss how the pre-existing chromatin landscape impacts DNA damage formation and guides DNA repair pathway choice, and how DNA damage-induced chromatin alterations control DNA damage signaling and repair, and DNA damage segregation through cell divisions. We also highlight that pathological alterations of histone proteins may trigger genome instability by impairing chromosome segregation and DNA repair, thus defining new oncogenic mechanisms and opening up therapeutic options.

A PRMT5-ZNF326 axis mediates innate immune activation upon replication stress.

DNA replication stress (RS) is a widespread phenomenon in carcinogenesis, causing genomic instability and extensive chromatin alterations. DNA damage leads to activation of innate immune signaling, but little is known about transcriptional regulators mediating such signaling upon RS. Using a chemical screen, we identified protein arginine methyltransferase 5 (PRMT5) as a key mediator of RS-dependent induction of interferon-stimulated genes (ISGs). This response is also associated with reactivation of endogenous retroviruses (ERVs). Using quantitative mass spectrometry, we identify proteins with PRMT5-dependent symmetric dimethylarginine (SDMA) modification induced upon RS. Among these, we show that PRMT5 targets and modulates the activity of ZNF326, a zinc finger protein essential for ISG response. Our data demonstrate a role for PRMT5-mediated SDMA in the context of RS-induced transcriptional induction, affecting physiological homeostasis and cancer therapy.

Microtubule-induced restriction of nuclear deformability in hypertrophic cardiomyopathy

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Leducq foundation Background Hypertrophic cardiomyopathy (HCM) is characterized by abnormal thickening of the left ventricular (LV) wall in conjunction with diastolic dysfunction with a prevalence of ~1:200. HCM pathophysiology is incompletely understood. We hypothesized that misregulated mechanotransduction plays an important role in the disease process. Extensive cytoskeletal remodeling has been observed in HCM patients, with a robust increase in tubulin and desmin levels, with a concomitant increase in stabilizing post-translational modifications, which we previously showed contributes to diastolic dysfunction. Microtubules are connected to the nucleus by the LINC complex. Little is known about the interaction of the non-sarcomeric cytoskeleton with the nucleus and the role this may have in the development of HCM. Purpose Relate cytoskeleton changes to nuclear and chromatin morphology in HCM patient myocardium and assess nuclear deformability in contracting cardiomyocytes with and without microtubule destabilizing drugs. Methods Nuclear morphology and chromatin organization was assessed using electron microscopy in cardiac septal tissue from 19 patients with obstructive HCM and compared to samples from 4 non-failing donors (NF). Further nuclear changes were studied using wild type (WT) and homozygous Mybpc3 c.2373InsG mice (Mybpc3c.2373InsG, KI), which have a HCM phenotype. Nuclear morphology was assessed in slices of 6 WT and 6 Mybpc3c.2373InsG paraffin-embedded mouse hearts. To study nuclear deformability during contractions, adult cardiomyocytes were isolated, stained with live cell dyes and imaged live while electrically paced. Lastly, DNA damaged was assessed in 6 WT and 6 Mybpc3c.2373InsG paraffin-embedded mouse hearts. Results Cardiomyocyte nuclei of HCM patients had a significantly higher number of invaginations (0.32±0.09 invaginations/µm in HCM versus 0.18±0.03 invaginations/µm in NF hearts), were more irregular of shape and had altered chromatin organization compared to those from NF hearts. Nuclei of Mybpc3c.2373InsG mice were considerably larger (322±75 µm³ versus 221±24 µm³). While sarcomere shortening is similar (7.0±1.4% in Mybpc3c.2373InsG versus 6.5±1.7% in WT), nuclei from Mybpc3c.2373InsG mice are less deformable than WT nuclei (figure). Addition of microtubule destabilizer nocodazole or detyrosination inhibitor EPO-Y restored nuclear deformability to WT levels, indicating a role of detyrosinated microtubules in nuclear deformability. In addition, nuclei from Mybpc3c.2373InsG have more extensive DNA damage (19.7±3.4 foci in Mybpc3c.2373InsG versus 9.1±3.2 foci in WT). Conclusion Extensive nuclear changes are observed in HCM patients and a HCM mouse model. Cytoskeletal remodeling results in less deformable nuclei that might contribute to chromatin alterations and increased DNA damage. Nuclear deformability can be restored by microtubule modifying drugs, specifically those that decrease microtubule detyrosination.

Exploring therapeutic approaches against Naegleria fowleri infections through the COVID box

Naegleria fowleri, known as the brain-eating amoeba, is the pathogen that causes the primary amoebic meningoencephalitis (PAM), a severe neurodegenerative disease with a fatality rate exceeding 95%. Moreover, PAM cases commonly involved previous activities in warm freshwater bodies that allow amoebae-containing water through the nasal passages. Hence, awareness among healthcare professionals and the general public are the key to contribute to a higher and faster number of diagnoses worldwide. Current treatment options for PAM, such as amphotericin B and miltefosine, are limited by potential cytotoxic effects. In this context, the repurposing of existing compounds has emerged as a promising strategy. In this study, the evaluation of the COVID Box which contains 160 compounds demonstrated significant in vitro amoebicidal activity against two type strains of N. fowleri. From these compounds, terconazole, clemastine, ABT-239 and PD-144418 showed a higher selectivity against the parasite compared to the remaining products. In addition, programmed cell death assays were conducted with these four compounds, unveiling compatible metabolic events in treated amoebae. These compounds exhibited chromatin condensation and alterations in cell membrane permeability, indicating their potential to induce programmed cell death. Assessment of mitochondrial membrane potential disruption and a significant reduction in ATP production emphasized the impact of these compounds on the mitochondria, with the identification of increased ROS production underscoring their potential as effective treatment options. This study emphasizes the potential of the mentioned COVID Box compounds against N. fowleri, providing a path for enhanced PAM therapies.

Exploring the Role of E6 and E7 Oncoproteins in Cervical Oncogenesis through MBD2/3-NuRD Complex Chromatin Remodeling.

Cervical cancer is among the highest-ranking types of cancer worldwide, with human papillomavirus (HPV) as the agent driving the malignant process. One aspect of the infection's evolution is given by epigenetic modifications, mainly DNA methylation and chromatin alteration. These processes are guided by several chromatin remodeling complexes, including NuRD. The purpose of this study was to evaluate the genome-wide binding patterns of the NuRD complex components (MBD2 and MBD3) in the presence of active HPV16 E6 and E7 oncogenes and to determine the potential of identified genes through an experimental model to differentiate between cervical precursor lesions, with the aim of establishing their utility as biomarkers. The experimental model was built using the CaSki cell line and shRNA for E6 and E7 HPV16 silencing, ChIP-seq, qRT-PCR, and Western blot analyses. Selected genes' expression was also assessed in patients. Several genes have been identified to exhibit altered transcriptional activity due to the influence of HPV16 E6/E7 viral oncogenes acting through the MBD2/MBD3 NuRD complex, linking them to viral infection and cervical oncogenesis. The impacted genes primarily play roles in governing gene transcription, mRNA processing, and regulation of translation. Understanding these mechanisms offers valuable insights into the process of HPV-induced oncogenesis.

Clinicopathologic Features and Cytologic Correlation of ALK-Rearranged Papillary Thyroid Carcinoma: A Series of Eight Cases

Anaplastic lymphoma kinase (ALK) gene fusions are rare in papillary thyroid carcinoma (PTC) but may serve as a therapeutic target. This study aims to evaluate the preoperative cytologic findings and clinicopathologic features of a series of eight ALK-rearranged PTCs from our pathology archives and consultations. All cases were confirmed by ALK D5F3 immunohistochemistry and six with additional targeted RNA-based next-generation sequencing (NGS). The original fine-needle aspiration (FNA) cytology diagnosis included the Bethesda System (TBS) category II in three (37.5%), TBS III in two (25%), TBS V in two (25%), and TBS VI in one (12.5%). Six cases had available FNA cytology and were reviewed. The cytologic features showed microfollicular architecture as well as limited or reduced nuclear elongation and chromatin alterations in all six. Nuclear grooves and pseudoinclusions were absent in two cases, rarely or focally noted in three, and frequently found in one. Two cases initially diagnosed as TBS II, showing microfollicular architecture without well-developed nuclear features, were revised to TBS III (with architectural atypia only). For histologic correlations, four were infiltrative follicular variant PTCs, three as classic subtype PTC with predominant follicular growth, and one as solid/trabecular subtype PTC. All eight cases demonstrated reduced PTC nuclear features with respect to nuclear elongation and chromatin alterations compared to those typically identified in “BRAF-like” PTCs. The NGS testing revealed EML4::ALK fusion in three, STRN::ALK fusion in two, and ITSN2::ALK fusion in one. In conclusion, although ALK-rearranged PTCs have been associated with neutral gene expression profile from a BRAF-RAS scoring perspective, the “RAS-like” nuclear features were more commonly identified in this series, resulting in frequent indeterminate diagnosis of preoperative FNA.

A critical role for Pol II CTD phosphorylation in heterochromatic gene activation

How gene activation works in heterochromatin, and how the mechanism might differ from the one used in euchromatin, has been largely unexplored. Previous work has shown that in SIR-regulated heterochromatin of Saccharomyces cerevisiae, gene activation occurs in the absence of covalent histone modifications and other alterations of chromatin commonly associated with transcription.Here we demonstrate that such activation occurs in a substantial fraction of cells, consistent with frequent transcriptional bursting, and this raises the possibility that an alternative activation pathway might be used. We address one such possibility, Pol II CTD phosphorylation, and explore this idea using a natural telomere-linked gene, YFR057w, as a model. Unlike covalent histone modifications, we find that Ser2, Ser5 and Ser7 CTD phosphorylated Pol II is prevalent at the drug-induced heterochromatic gene. Particularly enriched relative to the euchromatic state is Ser2 phosphorylation. Consistent with a functional role for Ser2P, YFR057w is negligibly activated in cells deficient in the Ser2 CTD kinases Ctk1 and Bur1 even though the gene is strongly stimulated when it is placed in a euchromatic context. Collectively, our results are consistent with a critical role for Ser2 CTD phosphorylation in driving Pol II recruitment and transcription of a natural heterochromatic gene – an activity that may supplant the need for histone epigenetic modifications.

201 Clear Cell Meningioma Tumorigenesis Is Characterized by Expression of Human Endogenous Retroviruses

INTRODUCTION: Human endogenous retroviruses (HERVs) are transcriptionally silenced genetic remnants of ancient exogenous retroviruses in differentiated cells yet critical to early tissue development. We recently found that aberrant expression of HERV-K envelope protein, caused by SMARCB1 mutation, is critical for tumorigenesis in atypical teratoid rhabdoid tumor (AT/RT). Clear cell meningioma (CCM) demonstrates analogous altered chromatin remodeling via SMARCE1 loss of function and syncytial histopathologic features thereby indicating a putative role for HERV expression in CCM tumorigenesis. METHODS: Eight CCM samples were evaluated for SMARCE1 status. We then quantified expression of HERV-K envelope and OCT-4 protein, a marker of pluripotency, by immunohistochemistry and compared them using linear regression. Active HERV transcript production was evaluated by RNA in-situ hybridization. Finally, we induced SMARCE1 loss in neural stem cells (NSC’s) and assessed expression of multiple HERV transcripts. We further modeled SMARCE1 knock-down in vivo in a chicken embryo model to evaluate timing of aberrant HERV expression and CCM development. RESULTS: All CCM tumor samples demonstrated loss of SMARCE1 and expression of HERV-K-Env and OCT-4 proteins and HERV transcripts. Expression was more robust in syncytial tumor-initiating cells and surrounding extracellular vesicles. HERV-K-Env expression positively correlated with OCT-4 expression (Pearson’s R = 0.7461, p = 0.033). Finally, SMARCE1 knock-down in NSC's and chicken embryos resulted in expression of multiple HERVs, supporting SMARCE1 loss of function as the mechanism of increased HERV expression in our CCM cohort. CONCLUSIONS: As mediated by loss of SMARCE1 function, HERV transcription allows for pathogenic exportation of HERV proteins to surrounding cells to maintain pluripotency in both in vitro and in vivo models. Aberrant HERV expression may therefore represent a targetable anticancer pathway for CCM and similar CNS tumors.

Effect of levetiracetam on DNA oxidation and glutathione content in a temporal lobe epilepsy model.

Levetiracetam (LEV) is a drug commonly used as an anticonvulsant. However, recent evidence points to a possible role as an antioxidant. We previously demonstrated the antioxidant properties of LEV by significantly increasing catalase and superoxide dismutase activities and decreasing the hydrogen peroxide (H2O2) levels in the hippocampus of rats with temporal lobe epilepsy (TLE) showing scavenging properties against the hydroxyl radical. The aim of the present work was to evaluate, the effect of LEV on DNA oxidation, by determining 8‑hydroxy‑2‑deoxyguanosine (8‑OHdG) levels, and glutathione content, through reduced (GSH) and oxidized (GSSG) glutathione levels, in the hippocampus of rats with TLE. Male Wistar rats were assigned to the control (CTRL), CTRL+LEV, epileptic (EPI) and EPI+LEV groups. TLE was induced using the lithium‑pilocarpine model. Thirteen weeks after TLE induction, LEV was administered for one week through osmotic pumps implanted subcutaneously. The determination of 8‑OHdG, GSH and GSSG levels were measured using spectrophotometric methods. We showed that LEV alone significantly increased 8‑OHdG and GSSG levels in the hippocampus of control rats compared to those in epileptic condition. No significant differences in GSH levels were observed. LEV could induce changes in the hippocampus increasing DNA oxidation and GSSG levels under nonepileptic condition but not protecting against the mitochondrial dysfunction observed in TLE probably by mechanisms related to changes in chromatin structure, neuroinflammation and alterations in redox components.