Melanoma antigen gene (MAGE) families are cancer-testis genes that normally show expression in the testes. However, their expressions have been linked with various types of human cancers, including BC. Therefore, the primary purposes of the present research were to assess the expression of MAGE-A, -B, and -C genes in Saudi female patients with BC and determine their regulation via the epigenetic mechanism. Ten BC samples were analyzed for the expression levels of nine MAGE-A genes, six MAGE-B genes, and three MAGE-C genes using the RT-PCR technique. All 18 evaluated genes except for MAGE-A1, -A3, -A4, and -B5 showed weak band expressions in some BC specimens. MAGE-A6 and -B2 were expressed in 40 % of the BC tissue samples, and MAGE-A9, -A10, and -B6 were expressed in 30 %. The lowest expression levels were found for MAGE-A11, -B1, -B3, -B4, -C1, and -C2 in 10 % of the BC specimens and for MAGE-A9,--B2, and --C3 in 20 % of the samples. The most frequently expressed gene was MAGE-A8 (found in 70 % of the BC samples), which suggests that it may serve as - a marker for screening of BC. In vitro treatment, the 5-aza-2\'-deoxycytidine agent led to a significant rise in mRNA expressions for all tested genes related to the MAGE-A family, except for MAGE-A10. By contrast, among the genes in the MAGE-B and -C families, only MAGE-B1 and -C2 exhibited detectable mRNA expression levels after treatment.

BACKGROUND: SETDB1 (SET domain bifurcated-1) is a histone H3-lysine 9 (H3K9)-specific methyltransferase that mediates heterochromatin formation and repression of target genes. Despite the assumed functional link between DNA methylation and SETDB1-mediated H3K9 trimethylations, several studies have shown that SETDB1 operates autonomously of DNA methylation in a region- and cell-specific manner. This study analyzes SETDB1-null HAP1 cells through a linked methylome and transcriptome analysis, intending to explore genes controlled by SETDB1-involved DNA methylation.
RESULTS: We investigated SETDB1-mediated regulation of DNA methylation and gene transcription in human HAP1 cells using reduced-representation bisulfite sequencing (RRBS) and RNA sequencing. While two-thirds of differentially methylated CpGs (DMCs) in genic regions were hypomethylated in SETDB1-null cells, we detected a plethora of C2H2-type zinc-finger protein genes (C2H2-ZFP, 223 of 749) among the DMC-associated genes. Most C2H2-ZFPs with DMCs in their promoters were found hypomethylated in SETDB1-KO cells, while other non-ZFP genes with promoter DMCs were not. These C2H2-ZFPs with DMCs in their promoters were significantly upregulated in SETDB1-KO cells. Similarly, C2H2-ZFP genes were upregulated in SETDB1-null 293T cells, suggesting that SETDB1\'s function in ZFP gene repression is widespread. There are several C2H2-ZFP gene clusters on chromosome 19, which were selectively hypomethylated in SETDB1-KO cells.
CONCLUSIONS: SETDB1 collectively and specifically represses a substantial fraction of the C2H2-ZFP gene family. Through the en-bloc silencing of a set of ZFP genes, SETDB1 may help establish a panel of ZFP proteins that are expressed cell-type specifically and thereby can serve as signature proteins for cellular identity.

Based on the impressive success of Car-T-cell therapy in the treatment of hematological malignancies, a broad application for solid tumors also appears promising. However, some important hurdles need to be overcome. One of these is certainly the identification of specific target antigens on cancer cells. Hypomethylation is a characteristic epigenetic aberration in many tumor entities. Genome-wide screenings for consistent DNA hypomethylations in tumors enable the identification of aberrantly upregulated transcripts, which might result in cell surface proteins. Thus, this approach provides a new perspective for the discovery of potential new Car-T-cell target antigens for almost every tumor entity. First, we focus on this approach as a possible treatment for prostate cancer.

Glioma is the most frequently diagnosed primary brain tumor and typically has a poor prognosis because of malignant proliferation and invasion. It is urgent to elucidate the mechanisms driving glioma tumorigenesis and develop novel treatments to address this deadly disease. Here, we first revealed that PDZK1 is expressed at high levels in gliomas. Promoter hypomethylation may cause high expression of PDZK1 in glioma. Knockdown of PDZK1 inhibits glioma cell proliferation and invasion in vitro. Mechanistically, further investigations revealed that the loss of PDZK1 expression by siRNA inhibited the activation of the AKT/mTOR signaling pathway, leading to cell cycle arrest and apoptosis. Clinically, high expression of PDZK1 predicts a poorer prognosis for glioma patients than low expression of PDZK1. Overall, our study revealed that PDZK1 acts as a novel oncogene in glioma by binding to AKT1 and maintaining the activation of the AKT/mTOR signaling pathway. Thus, PDZK1 may be a potential therapeutic target for glioma.

The 5-azacytidine (AZA) and decitabine (DEC) are noncytotoxic, differentiation-inducing therapies approved for treatment of myelodysplastic syndrome, acute myeloid leukemias (AML), and under evaluation as maintenance therapy for AML postallogeneic hematopoietic stem cell transplant and to treat hemoglobinapathies. Malignant cell cytoreduction is thought to occur by S-phase specific depletion of the key epigenetic regulator, DNA methyltransferase 1 (DNMT1) that, in the case of cancers, thereby releases terminal-differentiation programs. DNMT1-targeting can also elevate expression of immune function genes (HLA-DR, MICA, MICB) to stimulate graft versus leukemia effects. In vivo, there is a large inter-individual variability in DEC and 5-AZA activity because of pharmacogenetic factors, and an assay to quantify the molecular pharmacodynamic effect of DNMT1-depletion is a logical step toward individualized or personalized therapy. We developed and analytically validated a flow cytometric assay for DNMT1 epitope levels in blood and bone marrow cell subpopulations defined by immunophenotype and cell cycle state. Wild type (WT) and DNMT1 knock out (DKO) HC116 cells were used to select and optimize a highly specific DNMT1 monoclonal antibody. Methodologic validation of the assay consisted of cytometry and matching immunoblots of HC116-WT and -DKO cells and peripheral blood mononuclear cells; flow cytometry of H116-WT treated with DEC, and patient samples before and after treatment with 5-AZA. Analysis of patient samples demonstrated assay reproducibility, variation in patient DNMT1 levels prior to treatment, and DNMT1 depletion posttherapy. A flow-cytometry assay has been developed that in the research setting of clinical trials can inform studies of DEC or 5-AZA treatment to achieve targeted molecular pharmacodynamic effects and better understand treatment-resistance/failure.

Penile squamous cell carcinoma (SCC) is a rare and aggressive tumour mainly related to lifestyle behaviour and human papillomavirus (HPV) infection. Environmentally induced loss of imprinting (LOI) at the H19 differentially methylated region (H19DMR) is associated with many cancers in the early events of tumorigenesis and may be involved in the pathogenesis of penile SCC. We sought to evaluate the DNA methylation pattern at H19DMR and its association with HPV infection in men with penile SCC by bisulfite sequencing (bis-seq). We observed an average methylation of 32.2% ± 11.6% at the H19DMR of penile SCC and did not observe an association between the p16INK4a+ (p = 0.59) and high-risk HPV+ (p = 0.338) markers with methylation level. The average methylation did not change according to HPV positive for p16INK4a+ or hrHPV+ (35.4% ± 10%) and negative for both markers (32.4% ± 10.1%) groups. As the region analysed has a binding site for the CTCF protein, the hypomethylation at the surrounding CpG sites might alter its insulator function. In addition, there was a positive correlation between intense polymorphonuclear cell infiltration and hypomethylation at H19DMR (p = 0.035). Here, we report that hypomethylation at H19DMR in penile SCC might contribute to tumour progression and aggressiveness regardless of HPV infection.

Millions of people around the world are exposed to elevated levels of arsenic through food or drinking water. Epidemiological studies have linked chronic arsenic exposure to an increased risk of several cancers, cardiovascular disease, central nervous system neuropathies, and genotoxic as well as immunotoxic effects. In addition to the induction of oxidative stress and inhibition of DNA repair processes, epigenetic effects, including altered DNA methylation patterns resulting in aberrant gene expression, may contribute to carcinogenicity. However, the underlying mechanisms by which chronic micromolar concentrations of arsenite affect the methylation status of DNA are not fully understood. In this study, human HepG2 hepatocarcinoma cells were treated with 0.5-10 μM sodium arsenite for 24 h, 10, or 20 days. During these periods, the effects on global DNA methylation, cell cycle phase distribution, and gene expression were investigated. While no impact on DNA methylation was seen after short-term exposure, global hypomethylation was observed at both long-term exposure periods, with concomitant induction of the DNA methyltransferase genes DNMT1 and DNMT3B, while DNMT3A was slightly down-regulated. Pronounced time- and concentration-dependent effects were also seen in the case of genes involved in DNA damage response and repair, inflammation, oxidative stress response, and metal homeostasis. These results suggest that chronic low-dose arsenite exposure can lead to global hypomethylation. As an underlying mechanism, the consistent down-regulation of DNA methyltransferase genes could be excluded; alternatively, interactions at the protein level could play an important role.

Decitabine, a member of the 5-azanucleosides, has a dose-dependent mechanism of action in vitro: termination of DNA replication at high doses, and inhibition of DNA methyltransferase at low doses. The alteration of DNA methylation patterns by low-dose decitabine is hypothesized to upregulate genes, which promote myeloblast differentiation. In a phase III clinical trial, low-dose decitabine achieved a superior overall response rate (ORR) when compared with \'treatment choice\' [consisting of low-dose cytarabine (80%) and supportive care (20%)] as a frontline treatment for elderly patients with acute myeloid leukemia (AML). Despite an improved ORR, the median overall survival (OS) for elderly patients with AML was poor, <1 year. In turn, venetoclax was added to low-dose decitabine, the combination of which significantly improved the ORR and median OS in elderly patients with AML. Currently, hypomethylating agents are being combined with other novel therapies as investigational strategies for elderly and unfit patients with AML. They are also being evaluated as components of maintenance therapy in patients achieving remission. An oral formulation of decitabine has been developed which relies on the concomitant use of oral cedazuridine to protect against first pass metabolism. This oral formulation, which has been approved in myelodysplastic syndrome, is intended to increase convenience of use and therefore compliance in patients. This review characterizes the evolution of decitabine, its oral formulation, and its future in the treatment of AML.

BACKGROUND: Congenital heart defects (CHDs) affect approximately half of individuals with Down syndrome (DS), but the molecular reasons for incomplete penetrance are unknown. Previous studies have largely focused on identifying genetic risk factors associated with CHDs in individuals with DS, but comprehensive studies of the contribution of epigenetic marks are lacking. We aimed to identify and characterize DNA methylation differences from newborn dried blood spots (NDBS) of DS individuals with major CHDs compared to DS individuals without CHDs.
METHODS: We used the Illumina EPIC array and whole-genome bisulfite sequencing (WGBS) to quantitate DNA methylation for 86 NDBS samples from the California Biobank Program: (1) 45 DS-CHD (27 female, 18 male) and (2) 41 DS non-CHD (27 female, 14 male). We analyzed global CpG methylation and identified differentially methylated regions (DMRs) in DS-CHD versus DS non-CHD comparisons (both sex-combined and sex-stratified) corrected for sex, age of blood collection, and cell-type proportions. CHD DMRs were analyzed for enrichment in CpG and genic contexts, chromatin states, and histone modifications by genomic coordinates and for gene ontology enrichment by gene mapping. DMRs were also tested in a replication dataset and compared to methylation levels in DS versus typical development (TD) WGBS NDBS samples.
RESULTS: We found global CpG hypomethylation in DS-CHD males compared to DS non-CHD males, which was attributable to elevated levels of nucleated red blood cells and not seen in females. At a regional level, we identified 58, 341, and 3938 CHD-associated DMRs in the Sex Combined, Females Only, and Males Only groups, respectively, and used machine learning algorithms to select 19 Males Only loci that could distinguish CHD from non-CHD. DMRs in all comparisons were enriched for gene exons, CpG islands, and bivalent chromatin and mapped to genes enriched for terms related to cardiac and immune functions. Lastly, a greater percentage of CHD-associated DMRs than background regions were differentially methylated in DS versus TD samples.
CONCLUSIONS: A sex-specific signature of DNA methylation was detected in NDBS of DS-CHD compared to DS non-CHD individuals. This supports the hypothesis that epigenetics can reflect the variability of phenotypes in DS, particularly CHDs.

Hyperhomocysteinemia (HHcy) has been linked to development of Alzheimer\'s disease (AD) neuropathologically characterized by the accumulation of amyloid β (Aβ). Microglia (MG) play a crucial role in uptake of Aβ fibrils, and its dysfunction worsens AD. However, the effect of HHcy on MG Aβ phagocytosis remains unstudied.
We isolated MG from the cerebrum of HHcy mice with genetic cystathionine-β-synthase deficiency (Cbs-/-) and performed bulk RNA-seq. We performed meta-analysis over transcriptomes of Cbs-/- mouse MG, human and mouse AD MG, MG Aβ phagocytosis model, human AD methylome, and GWAS AD genes.
HHcy and hypomethylation conditions were identified in Cbs-/- mice. Through Cbs-/- MG transcriptome analysis, 353 MG DEGs were identified. Phagosome formation and integrin signaling pathways were found suppressed in Cbs-/- MG. By analyzing MG transcriptomes from 4 AD patient and 7 mouse AD datasets, 409 human and 777 mouse AD MG DEGs were identified, of which 37 were found common in both species. Through further combinatory analysis with transcriptome from MG Aβ phagocytosis model, we identified 130 functional-validated Aβ phagocytic AD MG DEGs (20 in human AD, 110 in mouse AD), which reflected a compensatory activation of Aβ phagocytosis. Interestingly, we identified 14 human Aβ phagocytic AD MG DEGs which represented impaired MG Aβ phagocytosis in human AD. Finally, through a cascade of meta-analysis of transcriptome of AD MG, functional phagocytosis, HHcy MG, and human AD brain methylome dataset, we identified 5 HHcy-suppressed phagocytic AD MG DEGs (Flt1, Calponin 3, Igf1, Cacna2d4, and Celsr) which were reported to regulate MG/MΦ migration and Aβ phagocytosis.
We established molecular signatures for a compensatory response of Aβ phagocytosis activation in human and mouse AD MG and impaired Aβ phagocytosis in human AD MG. Our discoveries suggested that hypomethylation may modulate HHcy-suppressed MG Aβ phagocytosis in AD.