DNA Methylation Of CpG Islands Research Articles

Abstract PO3-24-05: Cysteine metabolism related ferroptosis sensitivity in trastuzumab resistant HER2 positive breast cancer

Abstract Background Trastuzumab has shown great effectiveness in HER2 positive breast cancer treatment, but about 50% patients would undergo resistance during or after treatment. Although previous research has suggested several potential reasons for trastuzumab resistance, the metabolic reprogramming during resistance formation remains largely unclear. Here we identified aberrant ferroptosis associated cysteine metabolism in trastuzumab resistant HER2 positive breast cancer, which might become a novel target for overcoming resistance. Methods Trastuzumab sensitive HER2 positive breast cancer cell SKBR3 and resistant JITM1 were obtained for transcriptomics, proteomics, metabolomics and epigenomics analysis. Gene silencing was mediated by siRNAs. CUT&Tag was applied to compare H3K4me3 and H3K27me3 binding regions. DNA methylation levels and different methylated regions were evaluated by WGBS-seq. CRISPRi with dCas9-DNMT3A was applied to regulate specific DNA methylation in CpG islands. Lipid ROS was measured by flow cytometry with BODIPY-C11. Results Joint analyses of transcriptomics and proteomics according to ferroptosis pathways revealed downregulated glutathione metabolism, glutamate transmembrane and homocysteine metabolism processes, as well as upregulated fatty acid metabolism and iron metabolism pathways in JIMT1. Metabolomics verified that JIMT1 increased cysteine metabolism and decreased glutathione metabolism. SLC7A11 expression and GSH/GSSG ratio were increased in JIMT1, while no difference was observed in free cysteine. JIMT1 featured significant higher UGC codon usage bias and increased cysteinyl-tRNA synthetase. The abundance of H3K4me3 other than H3K27me3 in SLC7A11 promoter region was found increased in JIMT1, and the 5-mC level of CpG islands in SLC7A11 promoter region was shown decreased. Using dCas9-DNMT3A, the methylation of SLC7A11 promoter was enhanced and SLC7A11 expression was reduced in JIMT1. Inhibition of SLC7A11 by siRNAs, CRISPRi or Erastin all indicated a higher ferroptosis sensitivity in JIMT1. Conclusion Trastuzumab resistant HER2 positive breast cancer features aberrant cysteine metabolism resulting from altered H3K4me3 modification and DNA methylation in SLC7A11 promoter region. This might provide novel targets for further anti-HER2 treatment. Citation Format: Yongmei Yin, Yijia Hua, Ningjun Duan, Chunxiao Sun, Fan Yang. Cysteine metabolism related ferroptosis sensitivity in trastuzumab resistant HER2 positive breast cancer [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 PO3-24-05.

Roles of DNA Methylation in Color Alternation of Eastern Honey Bees (Apis cerana) Induced by the Royal Jelly of Western Honey Bees (Apis mellifera).

Honey bees have a very interesting phenomenon where the larval diets of two different honey bee species are exchanged, resulting in altered phenotypes, namely, a honey bee nutritional crossbreed. This is a classical epigenetic process, but its underlying mechanisms remain unclear. This study aims to investigate the contribution of DNA methylation to the phenotypic alternation of a Apis mellifera-Apis cerana nutritional crossbreed. We used a full nutritional crossbreed technique to rear A. cerana queens by feeding their larvae with A. mellifera royal-jelly-based diets in an incubator. Subsequently, we compared genome-wide methylation sequencing, body color, GC ratio, and the DMRs between the nutritional crossbreed, A. cerana queens (NQs), and control, A. cerana queens (CQs). Our results showed that the NQ's body color shifted to yellow compared to the black control queens. Genome methylation sequencing revealed that NQs had a much higher ratio of mCG than that of CQs. A total of 1020 DMGs were identified, of which 20 DMGs were enriched into key pathways for melanin synthesis, including tryptophan, tyrosine, dopamine, and phenylalanine KEGG pathways. Three key differentially methylated genes [OGDH, ALDH(NAD+) and ALDH7] showed a clear, altered DNA methylation in multiple CpG islands in NQs compared to CQs. Consequently, these findings revealed that DNA methylation participates in A. cerana-A. mellifera nutritional crossbreeding as an important epigenetic modification. This study serves as a model of cross-kingdom epigenetic mechanisms in insect body color induced by environmental factors.

Engineering CpG island DNA methylation in pluripotent cells through synthetic CpG-free ssDNA insertion

Cellular differentiation requires global changes to DNA methylation (DNAme), where it functions to regulate transcription factor, chromatin remodeling activity, and genome interpretation. Here, we describe a simple DNAme engineering approach in pluripotent stem cells (PSCs) that stably extends DNAme across target CpG islands (CGIs). Integration of synthetic CpG-free single-stranded DNA (ssDNA) induces a target CpG island methylation response (CIMR) in multiple PSC lines, Nt2d1 embryonal carcinoma cells, and mouse PSCs but not in highly methylated CpG island hypermethylator phenotype (CIMP)+ cancer lines. MLH1 CIMR DNAme spanned the CGI, was precisely maintained through cellular differentiation, suppressed MLH1 expression, and sensitized derived cardiomyocytes and thymic epithelial cells to cisplatin. Guidelines for CIMR editing are provided, and initial CIMR DNAme is characterized at TP53 and ONECUT1 CGIs. Collectively, this resource facilitates CpG island DNAme engineering in pluripotency and the genesis of novel epigenetic models of development and disease.

Transgenerational inheritance of acquired epigenetic signatures at CpG islands in mice

Transgenerational epigenetic inheritance in mammals remains a debated subject. Here, we demonstrate that DNA methylation of promoter-associated CpG islands (CGIs) can be transmitted from parents to their offspring in mice. We generated DNA methylation-edited mouse embryonic stem cells (ESCs), in which CGIs of two metabolism-related genes, the Ankyrin repeat domain 26 and the low-density lipoprotein receptor, were specifically methylated and silenced. DNA methylation-edited mice generated by microinjection of the methylated ESCs exhibited abnormal metabolic phenotypes. Acquired methylation of the targeted CGI and the phenotypic traits were maintained and transmitted across multiple generations. The heritable CGI methylation was subjected to reprogramming in parental PGCs and subsequently reestablished in the next generation at post-implantation stages. These observations provide a concrete step toward demonstrating transgenerational epigenetic inheritance in mammals, which may have implications in our understanding of evolutionary biology as well as the etiology, diagnosis, and prevention of non-genetically inherited human diseases.

Depletion of tet2 results in age-dependent changes in DNA methylation and gene expression in a zebrafish model of myelodysplastic syndrome.

Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic disorders characterized by ineffective hematopoiesis, cytopenias, and dysplasia. The gene encoding ten-eleven translocation 2 (tet2), a dioxygenase enzyme that catalyzes the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine, is a recurrently mutated tumor suppressor gene in MDS and other myeloid malignancies. Previously, we reported a stable zebrafish line with a loss-of-function mutation in the tet2 gene. The tet2m/m-mutant zebrafish developed a pre-MDS state with kidney marrow dysplasia, but normal circulating blood counts by 11 months of age and accompanying anemia, signifying the onset of MDS, by 24 months of age. In the current study, we collected progenitor cells from the kidney marrows of the adult tet2m/m and tet2wt/wt fish at 4 and 15 months of age and conducted enhanced reduced representation of bisulfite sequencing (ERRBS) and bulk RNA-seq to measure changes in DNA methylation and gene expression of hematopoietic stem and progenitor cells (HSPCs). A global increase in DNA methylation of gene promoter regions and CpG islands was observed in tet2m/m HSPCs at 4 months of age when compared with the wild type. Furthermore, hypermethylated genes were significantly enriched for targets of SUZ12 and the metal-response-element-binding transcription factor 2 (MTF2)-involved in the polycomb repressive complex 2 (PRC2). However, between 4 and 15 months of age, we observed a paradoxical global decrease in DNA methylation in tet2m/m HSPCs. Gene expression analyses identified upregulation of genes associated with mTORC1 signaling and interferon gamma and alpha responses in tet2m/m HSPCs at 4 months of age when compared with the wild type. Downregulated genes in HSPCs of tet2-mutant fish at 4 months of age were enriched for cell cycle regulation, heme metabolism, and interleukin 2 (IL2)/signal transducer and activator of transcription 5 (STAT5) signaling, possibly related to increased self-renewal and clonal advantage in HSPCs with tet2 loss of function. Finally, there was an overall inverse correlation between overall increased promoter methylation and gene expression.

DNA Methylation Analysis.

DNA methylation of promoter CpG islands silences their downstream genes, and enhancer methylation can be associated with decreased or increased gene expression. DNA methylation alterations in normal and diseased cells provide rich information, such as tissue origin, disease risk, patient response, and prognosis. DNA methylation status is detected by bisulfite conversion, which converts unmethylated cytosines into uracils but methylated cytosines very inefficiently. A genome-wide DNA methylation analysis is conducted by a BeadChip microarray or next-generation sequencing (NGS) of bisulfite-treated DNA. A region-specific DNA methylation analysis can be conducted by various methods, such as methylation-specific PCR (MSP), quantitative MSP, and bisulfite sequencing. This chapter provides protocols for bisulfite-mediated conversion, a BeadChip array-based method (Infinium), quantitative MSP, and bisulfite sequencing.

Tet Enzyme-Mediated Response in Environmental Stress and Stress-Related Psychiatric Diseases

Mental disorders caused by stress have become a worldwide public health problem. These mental disorders are often the results of a combination of genes and environment, in which epigenetic modifications play a crucial role. At present, the genetic and epigenetic mechanisms of mental disorders such as posttraumatic stress disorder or depression caused by environmental stress are not entirely clear. Although many epigenetic modifications affect gene regulation, the most well-known modification in eukaryotic cells is the DNA methylation of CpG islands. Stress causes changes in DNA methylation in the brain to participate in the neuronal function or mood-modulating behaviors, and these epigenetic modifications can be passed on to offspring. Ten-eleven translocation (Tet) enzymes are the 5-methylcytosine (5mC) hydroxylases of DNA, which recognize 5mC on the DNA sequence and oxidize it to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Tet regulates gene expression at the transcriptional level through the demethylation of DNA. This review will elaborate on the molecular mechanism and the functions of Tet enzymes in environmental stress-related disorders and discuss future research directions.

Epigenetic mechanism of L-type calcium channel β-subunit downregulation in short QT human induced pluripotent stem cell-derived cardiomyocytes with CACNB2 mutation.

A loss-of-function mutation in L-type calcium (Ca2+) channel subunit gene CACNB2 has been reported to cause short QT syndrome subtype 5 (SQT5). However, the mechanism underlying the loss-of-function of the Ca2+ channel has not been clarified. In the present study, we aim to explore the DNA methylation mechanism of L-type Ca2+ channel downregulation in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) of SQT5. The hiPSC-CMs were generated from a healthy donor and a SQT5 patient carrying the CACNB2 variant c.1439C > T/p.S480L. The variant was genetically corrected using ribonucleoprotein-based CRISPR/Cas9 technique to obtain an isogenic control cell line. The action potential (AP) and Ca2+ current were measured by patch clamp. Protein expression levels were determined by western blotting. Dot blotting and bisulfite sequence were performed for epigenetic study. Our results showed that AP durations at 10% repolarization (APD10) and 50% repolarization (APD50) were significantly shortened in SQT5 cells and both the expression level of the β-subunit and channel current of L-type Ca2+ channel were reduced. Besides, an increased level of whole-genome DNA methylation and DNA methylation of CpG island in the promoter region of CACNB2 gene was detected. Overexpression of demethylation enzyme could rescue the decreased expression of CACNB2 and the L-type Ca2+ current. In SQT5 hiPSC-CMs carrying the CACNB2-S480L variant, the decreased L-type Ca2+ current resulting from decreased CACNB2 protein expression was caused by enhanced methylation in the promoter region of the CACNB2 gene and upregulation of DNA methyltransferases might be one of the mechanisms.

Expression of GOT2 Is Epigenetically Regulated by DNA Methylation and Correlates with Immune Infiltrates in Clear-Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (KIRC) is the most common and highly malignant pathological type of kidney cancer, characterized by a profound metabolism dysregulation. As part of aspartate biosynthesis, mitochondrial GOT2 (glutamic-oxaloacetic transaminase 2) is essential for regulating cellular energy production and biosynthesis, linking multiple pathways. Nevertheless, the expression profile and prognostic significance of GOT2 in KIRC remain unclear. This study comprehensively analyzed the transcriptional levels, epigenetic regulation, correlation with immune infiltration, and prognosis of GOT2 in KIRC using rigorous bioinformatics analysis. We discovered that the expression levels of both mRNA and protein of GOT2 were remarkably decreased in KIRC tissues in comparison with normal tissues and were also significantly related to the clinical features and prognosis of KIRC. Remarkably, low GOT2 expression was positively associated with poorer overall survival (OS) and disease-free survival (DFS). Further analysis revealed that GOT2 downregulation is driven by DNA methylation in the promoter-related CpG islands. Finally, we also shed light on the influence of GOT2 expression in immune cell infiltration, suggesting that GOT2 may be a potential prognostic marker and therapeutic target for KIRC patients.

Association between CpG island DNA methylation in the promoter region of RELN and positive and negative types of schizophrenia.

ObjectiveTo explore the association between CpG island methylation in the promoter region of RELN and positive (type I) and negative (type II) types of schizophrenia, and investigate serum interleukin (IL)-1β, IL-6, and myelin basic protein (MBP) in schizophrenia.MethodsLevels of CpG island methylation in the promoter region of RELN were detected in peripheral blood of patients with schizophrenia (experimental group) and healthy individuals (control group), and serum IL-1β, IL-6, and MBP were measured.ResultsThe positive rate of CpG island methylation in the promoter region of RELN was higher in the experimental group than in the control group; however, there were no significant differences between type I and II patients. There were differences in Positive and Negative Syndrome Scale (PANSS) scores and serum IL-1β, IL-6, and MBP between type I and II patients. Furthermore, there were positive correlations between serum IL-1β, IL-6, and MBP and PANSS scores (negative symptoms) in type II patients.ConclusionCpG island methylation in the promoter region of RELN was associated with schizophrenia, but not with its clinical type. There may be different pathological mechanisms in type I and II schizophrenia, and type II schizophrenia may be associated with serum IL-1β, IL-6, and MBP.

Rescuing effects of periostin in advanced glycation end-products (AGEs) caused osteogenic and oxidative damage through AGE receptor mediation and DNA methylation of the CALCA promoter

An in vitro model was established to simulate a diabetes-type environment by treating human periodontal stem cells with advanced glycation end-products (AGEs). Periostin (POSTN) plays a crucial role in maintaining the integrity of periodontal tissues. However, the role of POSTN in human periodontal stem cells stimulated by AGEs remains unknown. Diabetes mellitus is considered a metabolic disease, and DNA methylation of CpG islands is a biomarker of metabolic syndromes. Diabetes has been found to be closely related to the DNA methylation of certain genes. Here, we investigated the protective mechanism and effect of POSTN on osteogenesis and oxidative stress in the AGE environment, and further explored the CpG island methylation of specific genes potentially mediated by POSTN. The optimal concentration of AGEs was screened using CCK8. AGEs were found to contribute to oxidative stress. Conversely, reactive oxygen species production and malondialdehyde and superoxide activity indicated that the AGE + POSTN group decreased oxidative injury. According to an alkaline phosphatase assay, Alizarin Red S staining, and the expression of key genes and proteins involved in osteogenesis, POSTN mitigated the inhibitory effects of AGE on cell proliferation and osteogenic differentiation potential during osteogenic differentiation. In contrast, the growth and osteogenesis of human periodontal stem cells were notably suppressed by POSTN knockdown. Bisulfite sequencing PCR was used to evaluate the DNA methylation status. Moreover, AGE elevated the expression of DNA methyltransferas 1 (DNMT1) and inhibited the activation of CALAL promoter methylation, which was rescued by the addition of POSTN and 5-Azacytidine (5-AZA). In conclusion, POSTN attenuated the AGE-induced inhibition of osteogenesis in periodontal ligament stem cells by reducing AGE receptor levels and DNA methylation of the calcitonin-related polypeptide α (CALCA) promoter. Thus, POSTN is a promising candidate for dental bone regeneration, representing a novel therapeutic agent for diabetic patients. The mechanism underlying these processes may provide new insights into novel therapeutic targets for improving abnormal bone metabolism in patients with diabetes.

Targeting GATA1 and p2x7r Locus Binding in Spinal Astrocytes Suppresses Chronic Visceral Pain by Promoting DNA Demethylation

Irritable bowel syndrome is a gastrointestinal disorder of unknown etiology characterized by widespread, chronic abdominal pain associated with altered bowel movements. Increasing amounts of evidence indicate that injury and inflammation during the neonatal period have long-term effects on tissue structure and function in the adult that may predispose to gastrointestinal diseases. In this study we aimed to investigate how the epigenetic regulation of DNA demethylation of the p2x7r locus guided by the transcription factor GATA binding protein 1 (GATA1) in spinal astrocytes affects chronic visceral pain in adult rats with neonatal colonic inflammation (NCI). The spinal GATA1 targeting to DNA demethylation of p2x7r locus in these rats was assessed by assessing GATA1 function with luciferase assay, chromatin immunoprecipitation, patch clamp, and interference in vitro and in vivo. In addition, a decoy oligodeoxynucleotide was designed and applied to determine the influence of GATA1 on the DNA methylation of a p2x7r CpG island. We showed that NCI caused the induction of GATA1, Ten-eleven translocation 3 (TET3), and purinergic receptors (P2X7Rs) in astrocytes of the spinal dorsal horn, and demonstrated that inhibiting these molecules markedly increased the pain threshold, inhibited the activation of astrocytes, and decreased the spinal sEPSC frequency. NCI also markedly demethylated the p2x7r locus in a manner dependent on the enhancement of both a GATA1–TET3 physical interaction and GATA1 binding at the p2x7r promoter. Importantly, we showed that demethylation of the p2x7r locus (and the attendant increase in P2X7R expression) was reversed upon knockdown of GATA1 or TET3 expression, and demonstrated that a decoy oligodeoxynucleotide that selectively blocked the GATA1 binding site increased the methylation of a CpG island in the p2x7r promoter. These results demonstrate that chronic visceral pain is mediated synergistically by GATA1 and TET3 via a DNA-demethylation mechanism that controls p2x7r transcription in spinal dorsal horn astrocytes, and provide a potential therapeutic strategy by targeting GATA1 and p2x7r locus binding.

Epigenetic Clock and Circadian Rhythms in Stem Cell Aging and Rejuvenation.

This review summarizes the current understanding of the interaction between circadian rhythms of gene expression and epigenetic clocks characterized by the specific profile of DNA methylation in CpG-islands which mirror the senescence of all somatic cells and stem cells in particular. Basic mechanisms of regulation for circadian genes CLOCK-BMAL1 as well as downstream clock-controlled genes (ССG) are also discussed here. It has been shown that circadian rhythms operate by the finely tuned regulation of transcription and rely on various epigenetic mechanisms including the activation of enhancers/suppressors, acetylation/deacetylation of histones and other proteins as well as DNA methylation. Overall, up to 20% of all genes expressed by the cell are subject to expression oscillations associated with circadian rhythms. Additionally included in the review is a brief list of genes involved in the regulation of circadian rhythms, along with genes important for cell aging, and oncogenesis. Eliminating some of them (for example, Sirt1) accelerates the aging process, while the overexpression of Sirt1, on the contrary, protects against age-related changes. Circadian regulators control a number of genes that activate the cell cycle (Wee1, c-Myc, p20, p21, and Cyclin D1) and regulate histone modification and DNA methylation. Approaches for determining the epigenetic age from methylation profiles across CpG islands in individual cells are described. DNA methylation, which characterizes the function of the epigenetic clock, appears to link together such key biological processes as regeneration and functioning of stem cells, aging and malignant transformation. Finally, the main features of adult stem cell aging in stem cell niches and current possibilities for modulating the epigenetic clock and stem cells rejuvenation as part of antiaging therapy are discussed.

DNA methylation mediated by melatonin was involved in ethylene signal transmission and ripening of tomato fruit

Melatonin (MT) as an evolutionarily highly conserved molecule plays an exceptional role in plants. In the present study, the regulation mechanism of the DNA methylation of CpG islands of ethylene signaling genes induced by MT on postharvest ripening of tomato fruit was studied. The ripening of tomato fruit was significantly promoted by the 0.5 mM MT treatment, as revealed by the appearance color, the lycopene, total soluble solids and vitamin C content of the fruit. In the MT treated fruit, the DNA methylation levels of CpG island of SlACS10 and SlERF-A1 were decreased, and the DNA methylation level of CpG island of SlCTR1 was increased. In addition, MT treatment increased the expression level of SlACS10, SlEIN3, SlERF-A1 and SlERT10, inhibited the expression level of SlCTR1, and by which the ethylene signaling might be activated and the ripening was promoted. The effect of MT on the DNA methylation of CpG islands of genes involved in ethylene signaling may contribute to the ripening of tomato fruit. The present study provided valuable information for understanding the essential role of DNA methylation in the postharvest ripening of tomato fruit.

Heat and Hypoxia Exposure Mediates Circadian Rhythms Response via Methylation Modification in Apostichopus japonicas

As global warming progresses, heat and hypoxia are gradually becoming important factors threatening the survival, reproduction, and development of marine organisms. To determine the effect of heat and hypoxia on Apostichopus japonicus, whole genome methylation of the respiratory tree was determined under heat, hypoxia, and heat-hypoxia conditions [designed as heat stress treatment (HT), hypoxia treatment (LO), and heat-hypoxia combined treatment (HL) groups]. The number of differentially methylated regions (DMRs) under three treatments was determined based on the Venn diagram. The network of the DMRs associated with promoters that were co-existed under the three conditions showed that circadian rhythm was involved based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Circadian rhythm-related genes, CRY1a, CRY1b, CLC, and TIM, decreased in LO and HL groups, while CRY1a, CRY1b, and BMAL1 increased in the HT group. Bisulfite sequencing PCR (BSP) showed that the methylation levels of CpG island regions in the promoters of CRY1a and CRY1b were upregulated in HT, LO, and HL groups, leading to the decreased promoter activity of CRY1a and CRY1b. RNAi of CRY1a and CRY1b led to increased enzyme activities of two energy-related enzymes, pyruvate kinase (PK) catalyzing the rate-limiting step in glycolysis, and ATPase hydrolyzing ATP to ADP, which were also increased under the three tested conditions. Thus, it was concluded that A. japonicus may respond to the heat, hypoxia, and heat-hypoxia stresses via the DNA methylation of heat, hypoxia, and heat-hypoxia stresses via the DNA methylation of CpG islands of circadian rhythm-related genes, which increased the activity of energy-related enzymes.

E2F6 initiates stable epigenetic silencing of germline genes during embryonic development

In mouse development, long-term silencing by CpG island DNA methylation is specifically targeted to germline genes; however, the molecular mechanisms of this specificity remain unclear. Here, we demonstrate that the transcription factor E2F6, a member of the polycomb repressive complex 1.6 (PRC1.6), is critical to target and initiate epigenetic silencing at germline genes in early embryogenesis. Genome-wide, E2F6 binds preferentially to CpG islands in embryonic cells. E2F6 cooperates with MGA to silence a subgroup of germline genes in mouse embryonic stem cells and in embryos, a function that critically depends on the E2F6 marked box domain. Inactivation of E2f6 leads to a failure to deposit CpG island DNA methylation at these genes during implantation. Furthermore, E2F6 is required to initiate epigenetic silencing in early embryonic cells but becomes dispensable for the maintenance in differentiated cells. Our findings elucidate the mechanisms of epigenetic targeting of germline genes and provide a paradigm for how transient repression signals by DNA-binding factors in early embryonic cells are translated into long-term epigenetic silencing during mouse development.

DNA Methylation of Human Choline Kinase Alpha Promoter-Associated CpG Islands in MCF-7 Cells.

Choline kinase (CK) is the enzyme catalyzing the first reaction in CDP-choline pathway for the biosynthesis of phosphatidylcholine. Higher expression of the α isozyme of CK has been implicated in carcinogenesis, and inhibition or downregulation of CKα (CHKA) is a promising anticancer approach. This study aimed to investigate the regulation of CKα expression by DNA methylation of the CpG islands found on the promoter of this gene in MCF-7 cells. Four CpG islands have been predicted in the 2000 bp promoter region of ckα (chka) gene. Six CpG island deletion mutants were constructed using PCR site-directed mutagenesis method and cloned into pGL4.10 vectors for promoter activity assays. Deletion of CpG4C region located between –225 and –56 significantly increased the promoter activity by 4-fold, indicating the presence of important repressive transcription factor binding site. The promoter activity of methylated full-length promoter was significantly lower than the methylated CpG4C deletion mutant by 16-fold. The results show that DNA methylation of CpG4C promotes the binding of the transcription factor that suppresses the promoter activity. Electrophoretic mobility shift assay analysis showed that cytosine methylation at MZF1 binding site in CpG4C increased the binding of putative MZF1 in nuclear extract. In conclusion, the results suggest that DNA methylation decreased the promoter activity by promoting the binding of putative MZF1 transcription factor at CpG4C region of the ckα gene promoter.

Methylation Patterns in the 5’ Flanking Region of The Hoxb9 Gene in Sea Turtle Embryos with Congenital Malformations

Sea turtles are wild animals whose embryonic development largely depends on environmental factors. The shell of sea turtles is a novelty among vertebrates and follows a Hox code and axial formula for the group. The hoxb9 gene is a transcription factor involved in the regulation of embryonic development, mainly during neural tube development and plays an important role in shaping the thoracic-lumbar transition of sea turtles. Although it is known that Hox genes are regulated by chromatin modulating proteins, DNA methylation in CpG islands may also contribute to gene regulation. Previous studies have shown a high prevalence of congenital malformations in the olive ridley sea turtle (Lepidochelys olivacea), including Schistosomus Reflexus (SR) syndrome. Here we studied the methylation profiles in the 5’ flanking region of hoxb9 comparing normal and abnormal sea turtle embryos. Our results indicate that methylation of the putative promoter region of hoxb9 in sea turtle embryos occurred, however methylated cytosines showed individual patterns, not directly related to the development of congenital malformations.

Methylation of ERβ 5'-untranslated region attenuates its inhibitory effect on ERα gene transcription and promotes the initiation and progression of papillary thyroid cancer.

Normal thyroid tissue displays a prevalent expression of ERβ than ERα, which drastically turns upside down in the initiation and progression of papillary thyroid cancer (PTC). The underlying molecular mechanism of this phenomenon remains unclear. Here, we demonstrated that ERα and ERβ were coexpressed in human thyroid tissues and cells. ERα mRNA (A-1) and ERβ mRNA (0N-1), transcribed from Promoter A of ERα gene and Promoter 0N of ERβ gene, respectively, were the major mRNA isoforms which mainly contributed to total ERα mRNA and total ERβ mRNA in human thyroid-derived cell lines and tissues. The expression levels of ERα mRNA (A-1) and total ERα mRNA were gradually increased, and those of ERβ mRNA (0N-1) and total ERβ mRNA were decreased by degree in the initiation and progression of PTC. No aberrant DNA methylation of ERα 5'-untranslated region was involved in its up-regulation; however, aberrant DNA methylation in Promoter 0N and Exon 0N of ERβ gene was found to be involved in its down-regulation in the initiation and progression of PTC. ERβ can repress ERα gene transcription via recruitment of NCoR and displacement of RNA polymerase II at the Sp1 site in ERα Promoter A-specific region in thyroid-derived cells. It is suggested that DNA methylation of CpG islands in Promoter 0N and Exon 0N of ERβ gene leads to a decreased ERβ gene expression, which attenuates its inhibitory effect on ERα gene transcription and results in an increased ERα gene expression, cell proliferation, initiation, and progression of PTC.

Alteration in stemness causes exclusivity between Epstein–Barr virus-positivity and microsatellite instability status in gastric cancer

Gastric cancer (GC) is a leading cause of cancer morbidity and mortality worldwide. This is due to the heterogeneous features of GC, which consist of a diverse molecular phenotype. Epstein-Barr virus (EBV)-positive GC and microsatellite instability (MSI)-high GC encompass similar epigenetic traits, including high levels of DNA methylation in CpG islands; however, EBV-positive and MSI-high GCs are mutually exclusive. We aimed to elucidate the underlying mechanism of this exclusivity. We knocked out MLH1 in EBV-positive GC cell lines SNU-719 and NCC24 via CRISPR-Cas9, and evaluated the modified cellular properties in vitro and in vivo. The MSI status of each cell line was screened with two marker capillary electrophoresis, and further diagnosed with five marker capillary electrophoresis and parallel sequencing using 21 markers. Initial evaluation showed that cell growth, migration, invasion, and MSI status were not affected by MLH1 silencing. However, with prolonged passage, GC cell lines gradually gained MSI and NCC24 cells were transformed to EBV-positive/MSI-high GC cells after 12months. Furthermore, MLH1 silencing reduced tumor stemness in SNU-719 and NCC24 regardless of the MSI status in vitro and in vivo. Our findings suggest that EBV-positivity and MSI-high status are mutually exclusive due to the immediate disadvantage in tumor stemness when MLH1 is silenced, whereas the establishment of MSI-high status in EBV-positive GCs required a longer period.