Loss of Y in regulatory T lymphocytes in the tumor micro-environment of primary colorectal cancers and liver metastases.

Male sex is a risk factor for colorectal cancer (CRC) with higher illness burden and earlier onset. Thus, we hypothesized that loss of chromosome Y (LOY) in the tumor micro-environment (TME) might be involved in oncogenesis. Previous studies show that LOY in circulating leukocytes of aging men was associated with shorter survival and non-hematological cancer, as well as higher LOY in CD4 + T-lymphocytes in men with prostate cancer vs. controls. However, nothing is known about LOY in leukocytes infiltrating TME and we address this aspect here. We studied frequency and functional effects of LOY in blood, TME and non-tumorous tissue. Regulatory T-lymphocytes (Tregs) in TME had the highest frequency of LOY (22%) in comparison to CD4 + T-lymphocytes and cytotoxic CD8 + T-lymphocytes. LOY score using scRNA-seq was also linked to higher expression of PDCD1, TIGIT and IKZF2 in Tregs. PDCD1 and TIGIT encode immune checkpoint receptors involved in the regulation of Tregs function. Our study sets the direction for further functional research regarding a probable role of LOY in intensifying features related to the suppressive phenotype of Tregs in TME and consequently a possible influence on immunotherapy response in CRC patients.

DMRTA2 supports glioma stem-cell mediated neovascularization in glioblastoma.

Glioblastoma (GBM) is the most common and lethal brain tumor in adults. Due to its fast proliferation, diffusive growth and therapy resistance survival times are less than two years for patients with IDH-wildtype GBM. GBM is noted for the considerable cellular heterogeneity, high stemness indices and abundance of the glioma stem-like cells known to support tumor progression, therapeutic resistance and recurrence. Doublesex- and mab-3-related transcription factor a2 (DMRTA2) is involved in maintaining neural progenitor cells (NPC) in the cell cycle and its overexpression suppresses NPC differentiation. Despite the reports showing that primary GBM originates from transformed neural stem/progenitors cells, the role of DMRTA2 in gliomagenesis has not been elucidated so far. Here we show the upregulation of DMRTA2 expression in malignant gliomas. Immunohistochemical staining showed the protein concentrated in small cells with high proliferative potential and cells localized around blood vessels, where it colocalizes with pericyte-specific markers. Knock-down of DMRTA2 in human glioma cells impairs proliferation but not viability of the cells, and affects the formation of the tumor spheres, as evidenced by strong decrease in the number and size of spheres in in vitro cultures. Moreover, the knockdown of DMRTA2 in glioma spheres affects the stabilization of the glioma stem-like cell-dependent tube formation in an in vitro angiogenesis assay. We conclude that DMRTA2 is a new player in gliomagenesis and tumor neovascularization and due to its high expression in malignant gliomas could be a biomarker and potential target for new therapeutic strategies in glioblastoma.

H2A.Z histone variants facilitate HDACi-dependent removal of H3.3K27M mutant protein in pediatric high-grade glioma cells.

Diffuse intrinsic pontine gliomas (DIPGs) are deadly pediatric brain tumors, non-resectable due to brainstem localization and diffusive growth. Over 80% of DIPGs harbor a mutation in histone 3 (H3.3 or H3.1) resulting in a lysine-to-methionine substitution (H3K27M). Patients with DIPG have a dismal prognosis with no effective therapy. We show that histone deacetylase (HDAC) inhibitors lead to a significant reduction in the H3.3K27M protein (up to 80%) in multiple glioma cell lines. We discover that the SB939-mediated H3.3K27M loss is partially blocked by a lysosomal inhibitor, chloroquine. The H3.3K27M loss is facilitated by co-occurrence of H2A.Z, as evidenced by the knockdown of H2A.Z isoforms. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis confirms the occupancy of H3.3K27M and H2A.Z at the same SB939-inducible genes. We discover a mechanism showing that HDAC inhibition in DIPG leads to pharmacological modulation of the oncogenic H3.3K27M protein levels. These findings show the possibility of directly targeting the H3.3K27M oncohistone.

Chromosomal positioning and epigenetic architecture influence DNA methylation patterns triggered by galactic cosmic radiation.

Despite surging interest in space travel in recent decades, the impacts of prolonged, elevated exposure to galactic cosmic radiation (GCR) on human health remain poorly understood. This form of ionizing radiation causes significant changes to biological systems including damage to DNA structure by altering epigenetic phenotype with emphasis on DNA methylation. Building on previous work by Kennedy et al. (Sci Rep 8(1): 6709. 10.1038/S41598-018-24755-8), we evaluated spatial DNA methylation patterns triggered by high-LET (56Fe, 28Si) and low-LET (X-ray) radiation and the influence of chromosome positioning and epigenetic architecture in distinct radial layers of cell nucleus. Next, we validated our results using gene expression data of mice irradiated with simulated GCR and JAXA astronauts. We showed that primarily 56Fe induces a persistent DNA methylation increase whereas 28Si and X-ray induce a decrease DNA methylation which is not persistent with time. Moreover, we highlighted the role of nuclear chromatin architecture in cell response to external radiation. In summary, our study provides novel insights towards epigenetic and transcriptomic response as well as chromatin multidimensional structure influence on galactic cosmic radiation damage.

Hypoxia-mediated regulation of DDX5 through decreased chromatin accessibility and post-translational targeting restricts R-loop accumulation.

Local hypoxia occurs in most solid tumors and is associated with aggressive disease and therapy resistance. Widespread changes in gene expression play a critical role in the biological response to hypoxia. However, most research has focused on hypoxia-inducible genes as opposed to those that are decreased in hypoxia. We demonstrate that chromatin accessibility is decreased in hypoxia, predominantly at gene promoters and specific pathways are impacted including DNA repair, splicing, and the R-loop interactome. One of the genes with decreased chromatin accessibility in hypoxia was DDX5, encoding the RNA helicase, DDX5, which showed reduced expression in various cancer cell lines in hypoxic conditions, tumor xenografts, and in patient samples with hypoxic tumors. Most interestingly, we found that when DDX5 is rescued in hypoxia, replication stress and R-loop levels accumulate further, demonstrating that hypoxia-mediated repression of DDX5 restricts R-loop accumulation. Together these data support the hypothesis that a critical part of the biological response to hypoxia is the repression of multiple R-loop processing factors; however, as shown for DDX5, their role is specific and distinct.

Personalized health risk assessment based on single-cell RNA sequencing analysis of a male with 45, X/48, XYYY karyotype.

Numeric sex chromosome abnormalities are commonly associated with an increased cancer risk. Here, we report a 14-year-old boy with a rare mosaic 45, X/48, XYYY karyotype presenting with subtle dysmorphic features and relative height deficiency, requiring growth hormone therapy. As only 12 postnatal cases have been described so far with very limited follow-up data, to assess the proband's long-term prognosis, including cancer risk, we performed high-throughput single-cell RNA sequencing (scRNA-seq) analysis. Although comprehensive cytogenetic analysis showed seemingly near perfect balance between 45, X and 48, XYYY cell populations, scRNA-seq revealed widespread differences in genotype distribution among immune cell fractions, specifically in monocytes, B- and T-cells. These results were confirmed at DNA level by digital-droplet PCR on flow-sorted immune cell types. Furthermore, deregulation of predominantly autosomal genes was observed, including TCL1A overexpression in 45, X B-lymphocytes and other known genes associated with hematological malignancies. Together with the standard hematological results, showing increased fractions of monocytes and CD4+/CD8+T lymphocytes ratio, long-term personalized hemato-oncological surveillance was recommended in the reported patient.

4-1BBL-containing leukemic extracellular vesicles promote immunosuppressive effector regulatory T cells.

Chronic and acute myeloid leukemia evade immune system surveillance and induce immunosuppression by expanding proleukemic Foxp3+ regulatory T cells (Tregs). High levels of immunosuppressive Tregs predict inferior response to chemotherapy, leukemia relapse, and shorter survival. However, mechanisms that promote Tregs in myeloid leukemias remain largely unexplored. Here, we identify leukemic extracellular vesicles (EVs) as drivers of effector proleukemic Tregs. Using mouse model of leukemia-like disease, we found that Rab27a-dependent secretion of leukemic EVs promoted leukemia engraftment, which was associated with higher abundance of activated, immunosuppressive Tregs. Leukemic EVs attenuated mTOR-S6 and activated STAT5 signaling, as well as evoked significant transcriptomic changes in Tregs. We further identified specific effector signature of Tregs promoted by leukemic EVs. Leukemic EVs-driven Tregs were characterized by elevated expression of effector/tumor Treg markers CD39, CCR8, CD30, TNFR2, CCR4, TIGIT, and IL21R and included 2 distinct effector Treg (eTreg) subsets: CD30+CCR8hiTNFR2hi eTreg1 and CD39+TIGIThi eTreg2. Finally, we showed that costimulatory ligand 4-1BBL/CD137L, shuttled by leukemic EVs, promoted suppressive activity and effector phenotype of Tregs by regulating expression of receptors such as CD30 and TNFR2. Collectively, our work highlights the role of leukemic extracellular vesicles in stimulation of immunosuppressive Tregs and leukemia growth. We postulate that targeting of Rab27a-dependent secretion of leukemic EVs may be a viable therapeutic approach in myeloid neoplasms.

Integration of single-cell RNA sequencing and spatial transcriptomics to reveal the glioblastoma heterogeneity.

Glioblastoma (GBM), a deadly brain tumor, is still one of a few lasting challenges of contemporary oncology. Current therapies fail to significantly improve patient survival due to GBM tremendous genetic, transcriptomic, immunological, and sex-dependent heterogeneity. Over the years, clinical differences between males and females were characterized. For instance, higher incidence of GBM in males or distinct responses to cancer chemotherapy and immunotherapy between males and females have been noted. Despite the introduction of single-cell RNA sequencing and spatial transcriptomics, these differences were not further investigated as studies were focused only on revealing the general picture of GBM heterogeneity. Hence, in this mini-review, we summarized the current state of knowledge on GBM heterogeneity revealed by single-cell RNA sequencing and spatial transcriptomics with regard to genetics, immunology, and sex-dependent differences. Additionally, we highlighted future research directions which would fill the gap of knowledge on the impact of patient's sex on the disease outcome.

Emerging Advances in Combinatorial Treatments of Epigenetically Altered Pediatric High-Grade H3K27M Gliomas.

Somatic mutations in histone encoding genes result in gross alterations in the epigenetic landscape. Diffuse intrinsic pontine glioma (DIPG) is a pediatric high-grade glioma (pHGG) and one of the most challenging cancers to treat, with only 1% surviving for 5 years. Due to the location in the brainstem, DIPGs are difficult to resect and rapidly turn into a fatal disease. Over 80% of DIPGs confer mutations in genes coding for histone 3 variants (H3.3 or H3.1/H3.2), with lysine to methionine substitution at position 27 (H3K27M). This results in a global decrease in H3K27 trimethylation, increased H3K27 acetylation, and widespread oncogenic changes in gene expression. Epigenetic modifying drugs emerge as promising candidates to treat DIPG, with histone deacetylase (HDAC) inhibitors taking the lead in preclinical and clinical studies. However, some data show the evolving resistance of DIPGs to the most studied HDAC inhibitor panobinostat and highlight the need to further investigate its mechanism of action. A new forceful line of research explores the simultaneous use of multiple inhibitors that could target epigenetically induced changes in DIPG chromatin and enhance the anticancer response of single agents. In this review, we summarize the therapeutic approaches against H3K27M-expressing pHGGs focused on targeting epigenetic dysregulation and highlight promising combinatorial drug treatments. We assessed the effectiveness of the epigenetic drugs that are already in clinical trials in pHGGs. The constantly expanding understanding of the epigenetic vulnerabilities of H3K27M-expressing pHGGs provides new tumor-specific targets, opens new possibilities of therapy, and gives hope to find a cure for this deadly disease.

Mapping chromatin accessibility and active regulatory elements reveals pathological mechanisms in human gliomas.

Chromatin structure and accessibility, and combinatorial binding of transcription factors to regulatory elements in genomic DNA control transcription. Genetic variations in genes encoding histones, epigenetics-related enzymes or modifiers affect chromatin structure/dynamics and result in alterations in gene expression contributing to cancer development or progression. Gliomas are brain tumors frequently associated with epigenetics-related gene deregulation. We perform whole-genome mapping of chromatin accessibility, histone modifications, DNA methylation patterns and transcriptome analysis simultaneously in multiple tumor samples to unravel epigenetic dysfunctions driving gliomagenesis. Based on the results of the integrative analysis of the acquired profiles, we create an atlas of active enhancers and promoters in benign and malignant gliomas. We explore these elements and intersect with Hi-C data to uncover molecular mechanisms instructing gene expression in gliomas.

Single-cell RNA sequencing reveals functional heterogeneity of glioma-associated brain macrophages.

Microglia are resident myeloid cells in the central nervous system (CNS) that control homeostasis and protect CNS from damage and infections. Microglia and peripheral myeloid cells accumulate and adapt tumor supporting roles in human glioblastomas that show prevalence in men. Cell heterogeneity and functional phenotypes of myeloid subpopulations in gliomas remain elusive. Here we show single-cell RNA sequencing (scRNA-seq) of CD11b+ myeloid cells in naïve and GL261 glioma-bearing mice that reveal distinct profiles of microglia, infiltrating monocytes/macrophages and CNS border-associated macrophages. We demonstrate an unforeseen molecular heterogeneity among myeloid cells in naïve and glioma-bearing brains, validate selected marker proteins and show distinct spatial distribution of identified subsets in experimental gliomas. We find higher expression of MHCII encoding genes in glioma-activated male microglia, which was corroborated in bulk and scRNA-seq data from human diffuse gliomas. Our data suggest that sex-specific gene expression in glioma-activated microglia may be relevant to the incidence and outcomes of glioma patients.

EGFR/FOXO3a/BIM signaling pathway determines chemosensitivity of BMP4-differentiated glioma stem cells to temozolomide.

Accumulating evidence suggests that glioma stem cells (GSCs), which are rare cells characterized by pluripotency and self-renewal ability, are responsible for glioblastoma (GBM) propagation, recurrence and resistance to therapies. Bone morphogenic proteins (BMPs) induce GSC differentiation, which leads to elimination of GSCs and sensitization of glioma to chemotherapeutics. Alterations in the epidermal growth factor receptor (EGFR) gene are detected in more than half of GBMs; however, the role of EGFR in the chemoresistance of GSCs remains unknown. Here, we examined whether EGFR signaling affects BMP4-induced differentiation of GSCs and their response to the alkylating drug temozolomide (TMZ). We show that BMP4 triggers the SMAD signaling cascade in GSCs independent of the EGFR level. BMP4 downregulated the levels of pluripotency markers (SOX2 and OLIG2) with a concomitant induction of an astrocytic marker (GFAP) and a neuronal marker (ß-Tubulin III). However, GSCs with different EGFR levels responded differently to treatments. BMP4-induced differentiation did not enhance sensitivity to TMZ in EGFRlow GSCs, in contrast to EGFRhigh GSCs, which underwent apoptosis. We then identified differences in cell cycle regulation. In EGFRlow cells, BMP4-triggered G1 cell cycle arrest which was not detected in EGFRhigh cells. RNA-seq profiles further highlighted transcriptomic alterations and distinct processes characterizing EGFR-dependent responses in the course of BMP4-induced differentiation. We found that the control of BIM (the pro-apoptotic BCL-2 family protein) by the AKT/FOXO3a axis only operated in BMP4-differentiated EGFRhigh cells upon TMZ treatment.

Search for novel STAT3-dependent genes reveals SERPINA3 as a new STAT3 target that regulates invasion of human melanoma cells.

Transcription factor signal transducer and activator of transcription 3 (STAT3) is constitutively activated in many cancers and promotes uncontrolled tumor growth and progression through multiple mechanisms. Compelling evidence shows tissue and cell-specific sets of STAT3 targets. Transcriptional targets of STAT3 in melanoma cells are largely unknown. Malignant melanoma is a deadly disease with highly aggressive and drug-resistant behavior. Less than 10% of patients with advanced melanomas reach the 5-year survival, partly due to the aggressive character of the tumor and ineffectiveness of current therapeutics for treating metastatic melanoma. STAT3 is constitutively activated in melanoma cells and plays important roles in its growth and angiogenesis in tumor xenograft studies. Moreover, highly metastatic melanoma cells have higher levels of active STAT3 than poorly metastatic ones. To identify genes that are driven by STAT3 in human melanoma cells, we performed JAK/STAT signaling specific and global gene expression profiling of human melanoma cells with silenced STAT3 expression. For selected genes, we performed computational identification of putative STAT3-binding sites and validated direct interactions STAT3 with defined promoters by using chromatin immunoprecipitation followed by qPCR. We found that STAT3 knockdown does not affect human melanoma cell viability, proliferation, or response to chemotherapeutics. We show that STAT3 regulates a discrete set of genes in melanoma cells, including SERPINA3, a novel STAT3 target gene, which is functionally involved in regulation of melanoma migration and invasion. Knockdown of STAT3 impaired cell migration and invasion, in part via regulation of its transcriptional target SERPINA3. Our results present novel targets and functions of STAT3 in melanoma cells.

In Search for Reliable Markers of Glioma-Induced Polarization of Microglia.

Immune cells accumulating in the microenvironment of malignant tumors are tumor educated and contribute to its growth, progression, and evasion of antitumor immune responses. Glioblastoma (GBM), the common and most malignant primary brain tumor in adults, shows considerable accumulation of resident microglia and peripheral macrophages, and their polarization into tumor-supporting cells. There are controversies regarding a functional phenotype of glioma-associated microglia/macrophages (GAMs) due to a lack of consistent markers. Previous categorization of GAM polarization toward the M2 phenotype has been found inaccurate because of oversimplification of highly complex and heterogeneous responses. In this study, we characterized functional responses and gene expression in mouse and human microglial cultures exposed to fresh conditioned media [glioma-conditioned medium (GCM)] from human U87 and LN18 glioma cells. Functional analyses revealed mutual communication reflected by strong stimulation of glioma invasion by microglial cells and increased microglial phagocytosis after GCM treatment. To define transcriptomic markers of GCM-activated microglia, we performed selected and global gene expression analyses of stimulated microglial cells. We found activated pathways associated with immune evasion and TGF signaling. We performed computational comparison of the expression patterns of GAMs from human GBMs and rodent experimental gliomas to select genes consistently changed in different datasets. The analyses of marker genes in GAMs from different experimental models and clinical samples revealed only a small set of common genes, which reflects variegated responses in clinical and experimental settings. Tgm2 and Gpnmb were the only two genes common in the analyzed data sets. We discuss potential sources of the observed differences and stress a great need for definitive elucidation of a functional state of GAMs.

Immune microenvironment of experimental rat C6 gliomas resembles human glioblastomas.

Glioblastoma (GBM) is the most aggressive primary brain tumor, with ineffective anti-tumor responses and a poor prognosis despite aggressive treatments. GBM immune microenvironment is heterogenous  and activation of specific immune populations in GBM is not fully characterized. Reliable animal models are critical for defining mechanisms of anti-tumor immunity. First we analyzed the immune subpopulations present in rat C6 gliomas. Using flow cytometry we determined kinetics of infiltration of myeloid cells and T lymphocytes into glioma-bearing brains. We found significant increases of the amoeboid, pro-tumorigenic microglia/macrophages, T helper (Th) and T regulatory (Treg) cells in tumor-bearing brains, and rare infiltrating T cytotoxic (Tc) cells. Transcriptomic analyses of glioma-bearing hemispheres revealed overexpression of invasion and immunosuppression-related genes, reflecting the immunosuppressive microenvironment. Microglia, sorted as CD11b+CD45low cells from gliomas, displayed the pro-invasive and immunosuppressive type of activation. Accumulation of Th and Treg cells combined with the reduced presence of Tc lymphocytes in rat gliomas may result in the lack of effective anti-tumor responses. Transcriptional profiles of CD11b+ cells and composition of immune infiltrates in C6 gliomas indicate that rat C6 gliomas employ similar immune system evasion strategies as human GBMs.

Environmental stimuli shape microglial plasticity in glioma.

In glioma, microglia and infiltrating macrophages are exposed to factors that force them to produce cytokines and chemokines, which contribute to tumor growth and to maintaining a pro-tumorigenic, immunosuppressed microenvironment. We demonstrate that housing glioma-bearing mice in enriched environment (EE) reverts the immunosuppressive phenotype of infiltrating myeloid cells, by modulating inflammatory gene expression. Under these conditions, the branching and patrolling activity of myeloid cells is increased, and their phagocytic activity is promoted. Modulation of gene expression depends on interferon-(IFN)-γ produced by natural killer (NK) cells. This modulation disappears in mice depleted of NK cells or lacking IFN-γ, and was mimicked by exogenous interleukin-15 (IL-15). Further, we describe a key role for brain-derived neurotrophic factor (BDNF) that is produced in the brain of mice housed in EE, in mediating the expression of IL-15 in CD11b+ cells. These data define novel mechanisms linking environmental cues to the acquisition of a pro-inflammatory, anti-tumor microenvironment in mouse brain.

SMARCB1-mediated SWI/SNF complex function is essential for enhancer regulation.

SMARCB1 (also known as SNF5, INI1, and BAF47), a core subunit of the SWI/SNF (BAF) chromatin-remodeling complex, is inactivated in nearly all pediatric rhabdoid tumors. These aggressive cancers are among the most genomically stable, suggesting an epigenetic mechanism by which SMARCB1 loss drives transformation. Here we show that, despite having indistinguishable mutational landscapes, human rhabdoid tumors exhibit distinct enhancer H3K27ac signatures, which identify remnants of differentiation programs. We show that SMARCB1 is required for the integrity of SWI/SNF complexes and that its loss alters enhancer targeting-markedly impairing SWI/SNF binding to typical enhancers, particularly those required for differentiation, while maintaining SWI/SNF binding at super-enhancers. We show that these retained super-enhancers are essential for rhabdoid tumor survival, including some that are shared by all subtypes, such as SPRY1, and other lineage-specific super-enhancers, such as SOX2 in brain-derived rhabdoid tumors. Taken together, our findings identify a new chromatin-based epigenetic mechanism underlying the tumor-suppressive activity of SMARCB1.

Down-regulation of IKKß expression in glioma-infiltrating microglia/macrophages is associated with defective inflammatory/immune gene responses in glioblastoma.

Glioblastoma (GBM) is an aggressive malignancy associated with profound host immunosuppression. Microglia and macrophages infiltrating GBM acquire the pro-tumorigenic, M2 phenotype and support tumor invasion, proliferation, survival, angiogenesis and block immune responses both locally and systematically. Mechanisms responsible for immunological deficits in GBM patients are poorly understood. We analyzed immune/inflammatory gene expression in five datasets of low and high grade gliomas, and performed Gene Ontology and signaling pathway analyses to identify defective transcriptional responses. The expression of many immune/inflammatory response and TLR signaling pathway genes was reduced in high grade gliomas compared to low grade gliomas. In particular, we found the reduced expression of the IKBKB, a gene coding for IKKß, which phosphorylates IκB proteins and represents a convergence point for most signal transduction pathways leading to NFκB activation. The reduced IKBKB expression and IKKß levels in GBM tissues were demonstrated by qPCR, Western blotting and immunohistochemistry. The IKKß expression was down-regulated in microglia/macrophages infiltrating glioblastoma. NFκB activation, prominent in microglia/macrophages infiltrating low grade gliomas, was reduced in microglia/macrophages in glioblastoma tissues. Down-regulation of IKBKB expression and NFκB signaling in microglia/macrophages infiltrating glioblastoma correlates with defective expression of immune/inflammatory genes and M2 polarization that may result in the global impairment of anti-tumor immune responses in glioblastoma.

Integration of genome-wide of Stat3 binding and epigenetic modification mapping with transcriptome reveals novel Stat3 target genes in glioma cells.

BACKGROUND: Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in many human tumors, including gliomas, and regulates the expression of genes implicated in proliferation, survival, apoptosis, angiogenesis and immune regulation. Only a small fraction of those genes has been proven to be direct STAT3 targets. In gliomas, STAT3 can play tumor suppressive or oncogenic roles depending on the tumor genetic background with target genes being largely unknown. RESULTS: We used chromatin immunoprecipitation, promoter microarrays and deep sequencing to assess the genome-wide occupancy of phospho (p)-Stat3 and epigenetic modifications of H3K4me3 and H3ac in C6 glioma cells. This combined assessment identified a list of 1200 genes whose promoters have both Stat3 binding sites and epigenetic marks characteristic for actively transcribed genes. The Stat3 and histone markings data were also intersected with a set of microarray data from C6 glioma cells after inhibition of Jak2/Stat3 signaling. Subsequently, we found 284 genes characterized by p-Stat3 occupancy, activating histone marks and transcriptional changes. Novel genes were screened for their potential involvement in oncogenesis, and the most interesting hits were verified by ChIP-PCR and STAT3 knockdown in human glioma cells. CONCLUSIONS: Non-random association between silent genes, histone marks and p-Stat3 binding near transcription start sites was observed, consistent with its repressive role in transcriptional regulation of target genes in glioma cells with specific genetic background.