CpG Islands In Regions Research Articles

Targeting microRNA methylation: Innovative approaches to diagnosing and treating hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) stands as the most prevalent form of primary liver cancer and is frequently linked to underlying chronic liver conditions such as hepatitis B, hepatitis C, and cirrhosis. Despite the progress achieved in the field of oncology, HCC remains a significant clinical challenge, primarily due to its typically late-stage diagnosis and the complex and multifaceted nature of its tumor biology. These factors contribute to the limited effectiveness of current treatment modalities and result in poor patient prognosis. Emerging research has underscored the vital role of microRNAs (miRNAs)-small, non-coding RNA molecules that play a pivotal part in the post-transcriptional regulation of gene expression. These miRNAs are integral to a wide array of cellular functions, including proliferation, apoptosis, and differentiation, and their dysregulation is closely associated with the pathogenesis of various cancers, notably HCC. A major focus in recent studies has been on the epigenetic regulation of miRNAs through methylation, a key mechanism that modulates gene expression. This process, involving the addition of methyl groups to CpG islands in the promoter regions of miRNA genes, can result in either gene silencing or activation, influencing the expression of oncogenes and tumor suppressor genes. Such alterations have profound implications for tumor growth, metastasis, and resistance to treatment. Evidence suggests that aberrant miRNA methylation can serve as a powerful biomarker for early detection and prognosis in HCC and may present novel opportunities for therapeutic intervention. This review aims to provide a comprehensive overview of the current landscape of miRNA methylation in HCC, elucidating its significance in the molecular mechanisms of liver cancer and examining its potential for clinical application. By exploring the diagnostic and therapeutic potential of miRNA methylation, we seek to highlight its value in enhancing personalized treatment strategies and improving patient outcomes.

High level of aneuploidy and recurrent loss of chromosome 11 as relevant features of somatotroph pituitary tumors

BackgroundSomatotroph neuroendocrine pituitary tumors (sPitNET) are a subtype of pituitary tumors that commonly cause acromegaly. Our study aimed to determine the spectrum of DNA copy number abnormalities (CNAs) in sPitNETs and their relevance.MethodsA landscape of CNAs in sPitNETs was determined using combined whole-genome approaches involving low-pass whole genome sequencing and SNP microarrays. Fluorescent in situ hybridization (FISH) was used for microscopic validation of CNAs. The tumors were also subjected to transcriptome and DNA methylation analyses with RNAseq and microarrays, respectively.ResultsWe observed a wide spectrum of cytogenetic changes ranging from multiple deletions, recurrent chromosome 11 loss, stable genomes, to duplication of the majority of the chromosomes. The identified CNAs were confirmed with FISH. sPitNETs with multiple duplications were characterized by intratumoral heterogeneity in chromosome number variation in individual tumor cells, as determined with FISH. These tumors were separate CNA-related sPitNET subtype in clustering analyses with CNA signature specific for whole genome doubling-related etiology. This subtype encompassed GNAS-wild type, mostly densely granulated tumors with favorable expression level of known prognosis-related genes, notably enriched with POUF1/NR5A1-double positive PitNETs. Chromosomal deletions in sPitNETs are functionally relevant. They occurred in gene-dense DNA regions and were related to genes downregulation and increased DNA methylation in the CpG island and promoter regions in the affected regions. Recurrent loss of chromosome 11 was reflected by lowered MEN1 and AIP. No such unequivocal relevance was found for chromosomal gains. Comparisons of transcriptomes of selected most cytogenetically stable sPitNETs with tumors with recurrent loss of chromosome 11 showed upregulation of processes related to gene dosage compensation mechanism in tumors with deletion. Comparison of stable tumors with those with multiple duplications showed upregulation of processes related to mitotic spindle, DNA repair, and chromatin organization. Both comparisons showed upregulation of the processes related to immune infiltration in cytogenetically stable tumors and deconvolution of DNA methylation data indicated a higher content of specified immune cells and lower tumor purity in these tumors.ConclusionssPitNETs fall into three relevant cytogenetic groups: highly aneuploid tumors characterized by known prognostically favorable features and low aneuploidy tumors including specific subtype with chromosome 11 loss.

Human stem cell-specific epigenetic signatures control transgene expression

Human stem cell-derived models have emerged as an important platform to study tissue differentiation and disease mechanisms. Those models could capitalize on biochemical and cell biological methodologies such as omics, autophagy, and organelle dynamics. However, epigenetic silencing in stem cells creates a barrier to apply genetically encoded tools. Here we investigate the molecular mechanisms underlying exogenously expressed gene silencing by employing multiple commonly used promoters in human induced pluripotent stem cells (iPSCs), glioblastoma cells (GBM), and embryonic kidney cells (HEK). We discover that all promoters tested are highly methylated on the CpG island regions with lower protein expression in iPSCs, as compared to non-iPSCs. Elongation factor 1 alpha short (EF1α short or EFS) promoter, which has fewer CpG island number compared to the other promoters, can drive relatively higher gene expression in iPSCs, despite CpG methylation. Adding a minimal A2 ubiquitous chromatin opening element (minimal A2 UCOE or miniUCOE) upstream of a promoter inhibits CpG methylation and enhances gene expression in iPSCs. Our results demonstrate stem cell type-specific epigenetic modification of transgenic promoter region and provide useful information for designing anti-silencing strategies to increase transgene expression in iPSCs.

MECP2 directly interacts with RNA polymerase II to modulate transcription in human neurons

Mutations in the methyl-DNA-binding protein MECP2 cause the neurodevelopmental disorder Rett syndrome (RTT). How MECP2 contributes to transcriptional regulation in normal and disease states is unresolved; it has been reported to be an activator and a repressor. We describe here the first integrated CUT&Tag, transcriptome, and proteome analyses using human neurons with wild-type (WT) and mutant MECP2 molecules. MECP2 occupies CpG-rich promoter-proximal regions in over four thousand genes in human neurons, including a plethora of autism risk genes, together with RNA polymerase II (RNA Pol II). MECP2 directly interacts with RNA Pol II, and genes occupied by both proteins showed reduced expression in neurons with MECP2 patient mutations. We conclude that MECP2 acts as a positive cofactor for RNA Pol II gene expression at many neuronal genes that harbor CpG islands in promoter-proximal regions and that RTT is due, in part, to the loss of gene activity of these genes in neurons.

Abstract LB293: O-GlcNAc transferase drives the proliferative state in adaptive drug tolerance

Abstract Drivers of the drug tolerant proliferative persister (DTPP) state have not been well investigated. Histone H3 lysine-4 trimethylation (H3K4me3), an active histone mark, might enable slow cycling drug tolerant persisters (DTP) to regain proliferative capacity. This study aimed to determine H3K4me3 transcriptionally active sites identifying a key regulator of DTPPs in melanoma. Deploying a model of adaptive cancer drug tolerance H3K4me3 ChIP-seq data of DTPPs guided identification of top transcription factor binding motifs. These suggested involvement of O-linked N-acetylglucosamine transferase (OGT), which was confirmed by metabolomics analysis and biochemical assays. OGT impact on DTPPs and adaptive resistance was explored in vitro and in vivo. H3K4me3 remodeling was widespread in CPG island regions and DNA binding motifs associated with O-GlcNAc marked chromatin. Accordingly, we observed an upregulation of OGT, O-GlcNAc and its binding partner TET1 in chronically treated cancer cells. Inhibition of OGT led to loss of H3K4me3 and downregulation of genes contributing to drug resistance. Genetic ablation of OGT prevented acquired drug resistance in vivo. Our findings uncover a fundamental mechanism of adaptive drug resistance that governs cancer cell reprogramming towards acquired drug resistance, a process that can be exploited to improve response duration and patient outcomes. Citation Format: Dinoop Ravindran Menon, Heinz Hammerlindl, Gregory Gimenez, Elmar Zuegner, Christoph Magens, Michael Eccles, Mayumi Fujita, Helmut Schaider. O-GlcNAc transferase drives the proliferative state in adaptive drug tolerance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB293.

Critical evaluation of the reliability of DNA methylation probes on the Illumina MethylationEPIC v1.0 BeadChip microarrays

ABSTRACT DNA methylation (DNAm) plays a crucial role in a number of complex diseases. However, the reliability of DNAm levels measured using Illumina arrays varies across different probes. Previous research primarily assessed probe reliability by comparing duplicate samples between the 450k-450k or 450k-EPIC platforms, with limited investigations on Illumina EPIC v1.0 arrays. We conducted a comprehensive assessment of the EPIC v1.0 array probe reliability using 69 blood DNA samples, each measured twice, generated by the Alzheimer’s Disease Neuroimaging Initiative study. We observed higher reliability in probes with average methylation beta values of 0.2 to 0.8, and lower reliability in type I probes or those within the promoter and CpG island regions. Importantly, we found that probe reliability has significant implications in the analyses of Epigenome-wide Association Studies (EWAS). Higher reliability is associated with more consistent effect sizes in different studies, the identification of differentially methylated regions (DMRs) and methylation quantitative trait locus (mQTLs), and significant correlations with downstream gene expression. Moreover, blood DNAm measurements obtained from probes with higher reliability are more likely to show concordance with brain DNAm measurements. Our findings, which provide crucial reliability information for probes on the EPIC v1.0 array, will serve as a valuable resource for future DNAm studies.

Methylation of microRNA genes and its effect on secondary xylem development of stem in poplar.

MicroRNAs (miRNAs) and DNA methylation are both vital regulators of gene expression. DNA methylation can affect the transcription of miRNAs, just like coding genes, through methylating the CpG islands in the gene regions of miRNAs. Although previous studies have shown that DNA methylation and miRNAs can each be involved in the process of wood formation, the relationship between the two has been relatively little studied in plant wood formation. Studies have shown that the second internode (IN2) (from top to bottom) of 3-month-old poplar trees can represent the primary stage of poplar stem development and IN8 can represent the secondary stage. There were also significant differences in DNA methylation patterns and miRNA expression patterns obtained from PS and SS. In this study, we first interactively analyzed methylation and miRNA sequencing data to identify 43 differentially expressed miRNAs regulated by differential methylation from the primary stage and secondary stage, which were found to be involved in multiple biological processes related to wood formation by enrichment analysis. In addition, six miRNA/target gene modules were finally identified as potentially involved in secondary xylem development of poplar stems through degradome sequencing and functional analysis. In conclusion, this study provides important reference information on the mechanism of interaction between different regulatory pathways of wood formation.

Abstract 3454: Cell-free DNA methylation and fragmentomic signatures identify tissue damage and predict cancer risk

Abstract Cell-free DNA (cfDNA) epigenetic and fragmentomic profiling has emerged as prominent non-invasive approaches for early cancer detection and subtyping. However, owing to difficulties in observing the early development of human malignancies, most cancer biomarker and evolution studies to date have primarily examined biologics following a diagnosis. Utilizing cfDNA as a screening tool for early disease detection requires assessment of blood plasma samples collected from asymptomatic individuals prior to the diagnosis of cancers. Here, we leverage the Ontario Health Study (OHS), a prospective longitudinal population cohort, to assess cfDNA profiles in blood plasma collected from participants that developed breast (n=476), prostate (n=342) and pancreatic (n=32) cancers throughout the study follow up time between 2009 and 2019. We performed cell-free methylated DNA immunoprecipitation and high-throughput sequencing (cfMeDIP-seq) on baseline plasma samples from incident cancer cases up to eight years prior to diagnosis, in addition to matched controls (n=404) with no history of cancer through follow-up. Genome-wide plasma cfDNA differential methylation analysis revealed significantly hypermethylated regions, particularly among CpG island and shore regions, were predictive of early breast cancers, and overlapped hypermethylated regions in solid cancer tissues relative to adjacent normal tissues and peripheral blood leukocytes. Using a repeated cross-validation strategy we found that as few as 200 hypermethylated regions can identify individuals with breast cancer in blood up to seven years prior to diagnosis among discovery set samples (AUC=0.725) and are highly generalizable to samples processed in separate batches (AUC = 0.650). Remarkably, integrating biomarker hypermethylated regions with fragmentomic features including fragment length and 5’ tetranucleotide motif proportions further increased accuracy for detecting individuals with early prostate cancers (AUC=0.804). In addition, we observed shared genome-wide cfDNA methylome changes associated with aging and heavy alcohol consumption in cfDNA. We identified increased shedding of tissue-specific methylation markers from liver and pancreatic tissue among individuals consuming more than three drinks a week relative to non-drinkers, reflective of potential signatures of tissue damage. Citation Format: Nicholas Cheng, Kimberly Skead, Tom Oullette, Scott Bratman, Daniel De Carvalho, David Soave, Philip Awadalla. Cell-free DNA methylation and fragmentomic signatures identify tissue damage and predict cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3454.

Abstract 3666: Multi-cancer early detection using metrics of DNA methylation based epigenetic instability

Abstract Cancers present significant changes in DNA methylation, which has proven to be highly useful in cell-free DNA (cfDNA) based cancer detection. Cancer epigenomes are marked by a high degree of intra- and inter-tumor epigenetic variation, indicative of the high level of epigenetic instability. However, the nature of the regions with high epigenetic instability and its utility as biomarker has not been explored. Here we developed a novel methodology and metric to measure the degree of epigenetic perturbation, termed the Epigenetic Instability Index (EII), for multiple cancer screening through cfDNA. Our novel methodology provides a sample specific score of epigenetic perturbation in relation to the expected normal state as opposed to only looking at CpG sites that change between samples. Through machine learning, we have elucidated 269 CpG-island regions which sufficiently capture the epigenetic instability in cancers. We have built classifier models using the EII metrics of these 269 regions and demonstrate that they can efficiently identify breast and lung cancer cases from cfDNA methylation data. Particularly, the models can differentiate even Stage IA of NSCLC with ~75% sensitivity at 95% specificity and early-stage breast cancer at ~68% sensitivity and 95% specificity. The EII metrics perform significantly better than metrics that measure absolute DNA methylation levels. Our studies highlight the potential of measuring epigenetic instability from cfDNA in any given sample in addition to traditional methods of methylation measurement for non-invasive screenings via liquid biopsies. Citation Format: Sara-Jayne Thursby, Zhicheng Jin, Steve B. Baylin, Malcolm Brock, Thomas Pisanic, Hariharan Easwaran. Multi-cancer early detection using metrics of DNA methylation based epigenetic instability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3666.

DIAGNOSTIC BIOMARKERS IN ORAL CANCER: CURRENT APPROACHES

Oral cancer is due to over expression of growth of abnormal cells which rapidly multiply and divide. oral cancer is the sixth most common tumour in the world and the highest mortality and morbidity rate amongst all the human malignancies. Betal nut, chewing pan and smokeless tobacco, intake of alcohol and chronic viral infections like human papilloma virus are some of the etiological factors of oral cancer. The pathogenies of oral cancers depend upon the interaction between the genetic, epigenetic and carcinogenetic factors and their association with tumour growth and the progression of cancer. Genetic alteration changes give rise to a multiplex of oral mucosal changes like cellular and architectural and dysplastic changes which help in grading of tumour (well-differentiated, moderately differentiated and poorly differentiated) oral cancer. K-67 and cyclin D1 are strong genetic changes taking place in oral malignancies. Diagnostic biomarkers are an essential tool for the early detection and better prognosis of oral cancer. There are invasive and non-invasive procedures for the diagnosis of oral cancer which include invasive procedures like biopsy and the non-invasive or minimally invasive procedures are blood, plasma, serum and saliva. The oral cancer biomarkers can be detected by taking the samples from cancer patients and running the molecular laboratory techniques like real time PCR for liquid biopsy, DNA sequencing, hybridization technique, single nucleotide polymorphism analysis and immunofluorescence for FFPS.
 Methylation includes hypermethylation and hypomethylation in which there is a defect in the promotor region of CPG island. The methylation process takes place in the early phase of the initiation of cancer so it is an important biomarker for the early detection of oral cancer. HOXA9, NID2, P16 AND PTEN are the genes of interest for hypermethylation in early cancer detection.
 Viral infection load like Epstein bar virus (EBV)and human papilloma virus (HPV) DNA load have specific biomarkers which can help in the early detection of oral cancer when combines with hypermethylation. Interferon and microRNA related gene are also upregulated in tumour and a biomarker for early detection of oral cancer.

DNA methylation in hearing-related genes in non-syndromic sensorineural hearing loss patients

BackgroundOur understanding of epigenetic modifications in the inner ear is very limited. Although epigenetic regulation of genes related to individual organ- and system-limited pathologies are generally expected to be tissue-specific, DNA methylation patterns in peripheral blood (PB) are found to be associated with the presence of several diseases with no typical hematological involvement. Here, we aimed to investigate whether there is a correlation between hearing-related genes’ promoter region methylation in the PB samples with the presence of non-syndromic sensorineural hearing loss (NSSHL) with an aim of future utilization of DNA methylation as biomarkers in hearing loss. The study included 26 patients with NSSHL and a control group of 20 healthy individuals. CpG islands in the promoter regions of the GJB-2, GJB-6, and SLC24A genes were analyzed using bisulfite sequencing, and methylation percentages were analyzed.ResultsMethylation levels at the 1st region of GJB-6 and the 1st and the 4th regions of SLC26A4 were found to differ significantly (p = 0.039, p = 0.042, and p = 0.029, respectively) between the patients and the control group. There was no statistically significant difference in methylation percentages of GJB-2 promoters. We also found that parents’ consanguinity determines the methylation levels in patients’ families.ConclusionsAccording to our knowledge, this is the first study that investigates epigenetic changes in the PB of patients with NSSHL. Despite the small sample size, our findings indicate that DNA methylation patterns in the PB could be of use for understanding epigenetic changes in the inner ear and the clinical management of NSSHL.

DNA methylation alterations in Lewy Body Dementia

AbstractBackgroundThe epigenetic mechanism of DNA methylation has been implicated in the pathophysiology of neurodegenerative disorders such as Lewy body dementia (LBD). DNA methylation profiling may help to identify novel treatment targets and early disease markers. We hypothesized that DNA methylation alterations are detectable in blood due the influence of genetic and environmental factors as well as immune system changes associated with LBD.MethodGenome‐wide DNA methylation profiling was carried out using reduced representation bisulfite sequencing in blood samples from 89 LBD patients and 67 controls. Fifty five LBD patients had amyloid PET imaging. Data processing was performed using FastQC, TrimGalore, Bismark and MethylKit. DNA methylation alterations at specific CpG sites as well as at promoter, CpG island and CpG shore regions were compared between LBD cases and controls. A log odds logistic regression was used to identify differential DNA methylation after adjusting for age, sex and smoking status. The false discovery rate (FDR) was used for correction for multiple testing. We also tested the diagnostic classification performance of the differentially methylated regions using receiver operating characteristic (ROC) curve analysis and examined the potential of DNA methylation as a predictor of amyloid PET positivity in LBD.ResultTwo single CpG sites at TNFRSF1B and EMC6 and three CpG shore regions at ABCG5, ETAA1 and TENM4 reached genome‐wide significance in the differential DNA methylation analysis comparing LBD cases and controls. The combined panel of the top three differentially methylated CpG shores reached an area under the curve (AUC) of 0.91 for the diagnosis of LBD compared to controls (sensitivity 0.89, specificity 0.85). DNA methylation at TENM4 was associated with amyloid PET positivity in LBD with an AUC of 0.84 (sensitivity 0.99, specificity 0.55).ConclusionOur data suggest that DNA methylation alterations in blood are observed in a cohort of LBD patients compared to controls. Our findings need to be replicated in larger cohorts and tested for specificity in LBD in order to explore their future potential as diagnostic markers and/or targets for novel therapeutics.

H3K4me3 remodeling induced acquired resistance through O-GlcNAc transferase.

Drivers of the drug tolerant proliferative persister (DTPP) state have not been well investigated. Histone H3 lysine-4 trimethylation (H3K4me3), an active histone mark, might enable slow cycling drug tolerant persisters (DTP) to regain proliferative capacity. This study aimed to determine H3K4me3 transcriptionally active sites identifying a key regulator of DTPPs. Deploying a model of adaptive cancer drug tolerance, H3K4me3 ChIP-Seq data of DTPPs guided identification of top transcription factor binding motifs. These suggested involvement of O-linked N-acetylglucosamine transferase (OGT), which was confirmed by metabolomics analysis and biochemical assays. OGT impact on DTPPs and adaptive resistance was explored in vitro and in vivo. H3K4me3 remodeling was widespread in CPG island regions and DNA binding motifs associated with O-GlcNAc marked chromatin. Accordingly, we observed an upregulation of OGT, O-GlcNAc and its binding partner TET1 in chronically treated cancer cells. Inhibition of OGT led to loss of H3K4me3 and downregulation of genes contributing to drug resistance. Genetic ablation of OGT prevented acquired drug resistance in in vivo models. Upstream of OGT, we identified AMPK as an actionable target. AMPK activation by acetyl salicylic acid downregulated OGT with similar effects on delaying acquired resistance. Our findings uncover a fundamental mechanism of adaptive drug resistance that governs cancer cell reprogramming towards acquired drug resistance, a process that can be exploited to improve response duration and patient outcomes.

Placental DNA methylation analysis of selective fetal growth restriction in monochorionic twins reveals aberrant methylated CYP11A1 gene for fetal growth restriction.

Fetal growth restriction (FGR) is associated with increased susceptibility to perinatal morbidity and mortality. Evidence suggests that epigenetic changes play critical roles in the regulation of fetal growth. We sought to present a comprehensive analysis of the associations between placental DNA methylation and selective fetal growth restriction (sFGR), which is a severe complication of monochorionic twin pregnancies, characterized by one fetus experiencing restricted growth. Genome-wide methylation analysis was performed on 24 placental samples obtained from 12 monochorionic twins with sFGR (Cohort 1) using Illumina Infinium MethylationEPIC BeadChip. Integrative analysis of our EPIC data and two previous placental methylation studies of sFGR (a total of 30 placental samples from 15 sFGR twins) was used to identify convincing differential promoter methylation. Validation analysis was performed on the placentas from 15 sFGR twins (30 placental samples), 15 FGR singletons, and 14 control singletons (Cohort 2) using pyrosequencing, quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry (IHC). A globe shift toward hypomethylation was identified in the placentas of growth-restricted fetuses compared with the placentas of normal fetuses in monochorionic twins, including 5625 hypomethylated CpGs and 452 hypermethylated CpGs, especially in the regions of CpG islands, gene-body and promoters. The analysis of pathways revealed dysregulation primarily in steroid hormone biosynthesis, metabolism, cell adhesion, signaling transduction, and immune response. Integrative analysis revealed a differentially methylated promoter region in the CYP11A1 gene, encoding a rate-limiting enzyme of steroidogenesis converting cholesterol to pregnenolone. The CYP11A1 gene was validated to have hypomethylation and higher mRNA expression in sFGR twins and FGR singletons. In conclusion, our findings suggested that the changes in placental DNA methylation pattern in sFGR may have functional implications for differentially methylated genes and regulatory regions. The study provides reliable evidence for identifying abnormally methylated CYP11A1 gene in the placenta of sFGR.

PTEN regulates expression of its pseudogene in glioblastoma cells in DNA methylation-dependent manner

Glioblastoma (GBM) is the most aggressive and frequent type of primary brain cancer in adult patients. One of the key molecular features associated with GBM pathogenesis is the dysfunction of PTEN oncosuppressor. In addition to PTEN gene, humans and several primates possess processed PTEN pseudogene (PTENP1) that gives rise to long non-coding RNA lncPTENP1-S. Regulation and functions of PTEN and PTENP1 are highly interconnected, however, the exact molecular mechanism of how these two genes affect each other remains unclear. Here, we analyzed the methylation level of the CpG islands (CpGIs) in the promoter regions of PTEN and PTENP1 in patient-derived GBM neurospheres. We found that increased PTEN methylation corelates with decreased PTEN mRNA level. Unexpectedly, we showed the opposite trend for PTENP1. Using targeted methylation and demethylation of PTENP1 CpGI, we demonstrated that DNA methylation increases lncPTENP1-S expression in the presence of wild type PTEN protein but decreases lncPTENP1-S expression if PTEN protein is absent. Further experiments revealed that PTEN protein binds to PTENP1 promoter region and inhibits lncPTENP1-S expression if its CpGI is demethylated. Interestingly, we did not detect any effect of lncPTENP1-S on the level of PTEN mRNA, indicating that in GBM cells PTENP1 is a downstream target of PTEN rather than its upstream regulator. Finally, we studied the functions of lncPTENP1-S and demonstrated that it plays a pro-oncogenic role in GBM cells by upregulating the expression of cancer stem cell markers and decreasing cell adhesion.

Transcriptional repression of lncRNA and miRNA subsets mediated by LRF during erythropoiesis

Non-coding RNA (ncRNA) species, mainly long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) have been currently imputed for lesser or greater involvement in human erythropoiesis. These RNA subsets operate within a complex circuit with other epigenetic components and transcription factors (TF) affecting chromatin remodeling during cell differentiation. Lymphoma/leukemia-related (LRF) TF exerts higher occupancy on DNA CpG rich sites and is implicated in several differentiation cell pathways and erythropoiesis among them and also directs the epigenetic regulation of hemoglobin transversion from fetal (HbF) to adult (HbA) form by intervening in the γ-globin gene repression. We intended to investigate LRF activity in the evolving landscape of cells’ commitment to the erythroid lineage and specifically during HbF to HbA transversion, to qualify this TF as potential repressor of lncRNAs and miRNAs. Transgenic human erythroleukemia cells, overexpressing LRF and further induced to erythropoiesis, were subjected to expression analysis in high LRF occupancy genetic loci-producing lncRNAs. LRF abundance in genetic loci transcribing for studied lncRNAs was determined by ChIP-Seq data analysis. qPCRs were performed to examine lncRNA expression status. Differentially expressed miRNA pre- and post-erythropoiesis induction were assessed by next-generation sequencing (NGS), and their promoter regions were charted. Expression levels of lncRNAs were correlated with DNA methylation status of flanked CpG islands, and contingent co-regulation of hosted miRNAs was considered. LRF-binding sites were overrepresented in LRF overexpressing cell clones during erythropoiesis induction and exerted a significant suppressive effect towards lncRNAs and miRNA collections. Based on present data interpretation, LRF’s multiplied binding capacity across genome is suggested to be transient and associated with higher levels of DNA methylation.Key messagesDuring erythropoiesis, LRF displays extensive occupancy across genetic loci.LRF significantly represses subsets of lncRNAs and miRNAs during erythropoiesis.Promoter region CpG islands’ methylation levels affect lncRNA expression.MiRNAs embedded within lncRNA loci show differential regulation of expression.

PD-1 Overexpression in Sézary Syndrome Is Epigenetically Regulated

PD-1 Overexpression in Sézary Syndrome Is Epigenetically Regulated

Correlation of DNA methylation and lymph node metastasis in papillary thyroid carcinoma.

Papillary thyroid carcinoma (PTC) is the most common type of thyroid cancer with a primarily good prognosis, and its 10-year survival rate is over 90%. However, PTC is prone to early lymph node metastasis. Thyroid cancer tissues from PTC patients with lymphatic metastasis and normal tissues were collected for DNA methylation analysis. Different methylation sites, different methylation regions, gene-enriched pathways, and protein-protein interactions (PPIs) were analyzed. There were 1004 differentially methylated sites in the PTC group versus the control group; these involved 479 hypermethylated sites in 415 related genes, 525 hypomethylated sites in 482 related genes, 64 differentially methylated regions located in the CpG island region, 34 differentially methylated genes closely related to thyroid cancer, and 17 genes with differentially methylated genes in the DNA promoter region. NDRG4 hypermethylation and FOXO3, ZEB2, and CDK6 hypomethylation were associated with PTC lymph node metastasis.

Abstract B013: Epigenetic clocks and breast cancer outcomes: A scoping review. [R

Abstract Introduction: Age is a strong predictor for breast cancer (BCa) risk. The median age at diagnosis varies by race/ethnicity. In the United States (U.S.), non-White women are more likely to be diagnosed at a younger age than non-Hispanic White (NHW) women. Epigenetic age, measured as changes to DNA via methylation (DNAm) using epigentic clocks, is highly accurate at estimating cellular age using several tissues and cell types may differ from chronological age. Epigenetic age is more robust than other molecular markers of aging, such as telomere length. Differences between epigenetic age and chronological age, termed epigenetic age acceleration, may be the driver of age and race/ethnicity discrepancies when estimating BCa risk. Age-related methylation between disease-free and tumor breast tissue is significantly differentially methylated. For example, aberrant methylation of promoter regions of polycomb group protein target genes and CpG islands associated with BCa risk and progression. Therefore, the purpose of this scoping review is to gather data on epigenetic clocks as a predictor for BCa incidence. Study objective: Synthesize the existing evidence on epigenetic accelerated aging measured with epigenetic clocks and breast cancer risk in the U.S. Methods: The PROSPERO Scoping Review Protocol was used for this project. We performed multiple PubMed searches utilizing search language developed by an experienced search librarian. The searches were conducted from January 2022 to April 2022. The searches yielded a total of 2,908 studies in the search process. The eligibility for inclusion applied during article review was: 1) the study must have used an epigenetic clock to assess epigenetic accelerated age in their sample, 2) the study’s primary outcome needed to be breast cancer risk, 3) the study needed to use a U.S. based study sample 3) the study needed to be published in a peer-reviewed PubMed indexed journal, 4) the study must have been published by April 30, 2022. Results: A total of seven articles from the 2,908 were determined eligible for inclusion. Data extraction from these seven articles demonstrated epigenetic accelerated age measured with epigenetic clocks does have an associative relationship with the development of breast cancer. Discussion: BCa risk is also known to drastically increase after menopause, although there are significant differences of menopausal transition in race/ethnicity. On average, African American, Asian, and Latina women begin menopause earlier than non-Hispanic White women. Emerging evidence suggests that earlier menopause in these populations may be a result of structural factors experienced by historically disadvantaged communities. The studies included in this review focused primarily on NHW women. More research is needed to better understand the role of DNAm and accelerated epigenetic age in diverse populations. Citation Format: Jennifer Daw, Devin Saunders, Adria Vasquez, Celina Valencia. Epigenetic clocks and breast cancer outcomes: A scoping review. [R] [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr B013.

Methylation status of hypothalamic Mkrn3 promoter across puberty.

Makorin RING finger protein 3 (MKRN3) is an important factor located on chromosome 15 in the imprinting region associated with Prader-Willi syndrome. Imprinted MKRN3 is expressed in hypothalamic regions essential for the onset of puberty and mutations in the gene have been found in patients with central precocious puberty. The pubertal process is largely controlled by epigenetic mechanisms that include, among other things, DNA methylation at CpG dinucleotides of puberty-related genes. In the present study, we investigated the methylation status of the Mkrn3 promoter in the hypothalamus of the female mouse before, during and after puberty. Initially, we mapped the 32 CpG dinucleotides in the promoter, the 5'UTR and the first 50 nucleotides of the coding region of the Mkrn3 gene. Moreover, we identified a short CpG island region (CpG islet) located within the promoter. Methylation analysis using bisulfite sequencing revealed that CpG dinucleotides were methylated regardless of developmental stage, with the lowest levels of methylation being found within the CpG islet region. In addition, the CpG islet region showed significantly lower methylation levels at the pre-pubertal stage when compared with the pubertal or post-pubertal stage. Finally, in silico analysis of transcription factor binding sites on the Mkrn3 CpG islet identified the recruitment of 29 transcriptional regulators of which 14 were transcriptional repressors. Our findings demonstrate the characterization and differential methylation of the CpG dinucleotides located in the Mkrn3 promoter that could influence the transcriptional activity in pre-pubertal compared to pubertal or post-pubertal period. Further studies are needed to clarify the possible mechanisms and effects of differential methylation of the Mkrn3 promoter.