Detection of LINE-1 hypomethylation in cfDNA of Esophageal Adenocarcinoma Patients

DNA methylation plays an important role in cancer development. Cancer cells exhibit two types of DNA methylation alteration: site-specific hypermethylation at promoter of oncosuppressor genes and global DNA hypomethylation. This study evaluated the methylation patterns of long interspersed nuclear element (LINE-1) sequences which, due to their relative abundance in the genome, are considered a good surrogate indicator of global DNA methylation. LINE-1 methylation status was investigated in the cell-free DNA (cfDNA) of 21 patients, 19 with esophageal adenocarcinoma (EADC) and 2 with Barrett’s esophagus (BE). The two BE patients and one EADC patient were also analyzed longitudinally. Methylation status was analyzed using restriction enzymes and DNA amplification. This methodology was chosen to avoid bisulfite conversion, which we considered inadequate for cfDNA analysis. Indeed, cfDNA is characterized by poor quality and low concentration, and bisulfite conversion might worsen these conditions. Results showed that hypomethylated LINE-1 sequences are present in EADC cfDNA. Furthermore, longitudinal studies in BE suggested a correlation between methylation status of LINE-1 sequences in cfDNA and progression to EADC. In conclusion, our study indicated the feasibility of our methodological approach to detect hypomethylation events in cfDNA from EADC patients, and suggests LINE-1 methylation analysis as a new possible molecular assay to integrate into patient monitoring.

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268 LIQUID BIOPSY IN ESOPHAGEAL ADENOCARCINOMA: ROLE OF LINE-1 HYPOMETHYLATION

Diseases of the Esophagus ◽

10.1093/dote/doaa087.54 ◽

2020 ◽

Vol 33 (Supplement_1) ◽

Author(s):

R Alfieri ◽

E Boldrin ◽

M Mazza ◽

S Realdon ◽

M Fassan ◽

...

Keyword(s):

Esophageal Adenocarcinoma ◽

Methodological Approach ◽

Methylation Status ◽

Neoadjuvant Treatment ◽

Dna Amplification ◽

Restriction Enzymes ◽

Clinical Staging ◽

Low Grade ◽

The Difference ◽

Methylation Patterns

Abstract Esophageal Adenocarcinoma (EADC) prognosis is still poor. Clinical staging doesn’t allow accurate prediction of response to neoadjuvant treatment and survival. Molecular characterization of EADC is an expanding field aimed to predict treatment response and prognosis. Hypomethylation of Long Interspersed Nuclear Element 1 (LINE-1) sequences recently emerged as a frequent epigenetic alteration in EADC. Aim of the study was to evaluate methylation patterns of LINE-1 that is considered a good surrogate of global DNA methylation. Methods We investigated cell free DNA (cfDNA) of 21 patients, 19 with EADC and 2 with Barrett’s esophagus (BE). To verify the possibility to use LINE-1 methylation status to predict tumor behaviour, we extended the analysis to a few longitudinal cases, one EADC and two BE. One BE patient progressed to low-grade dysplasia (LGD) and high-grade dysplasia (HGD)/EADC during surveillance, while the other remains stable. LINE-1 promoter methylation was analyzed using restriction enzymes and DNA amplification. Results Using this approach, we were able to detect the presence of hypomethylated LINE-1 sequences in EADC cfDNA. The difference in methylation level between cfDNA and constitutive DNA was statistically significant (p = 0.0001). In one EADC patient, treated with esophagectomy, LINE-1 hypomethylation was found at lower level after resection and at higher level at recurrence. In another patient affected by BE, LINE-1 hypomethylation increased during the progression from metaplastic epithelium to EADC, switching back to the level of constitutive DNA after endoscopic resection. The third patient, with stable BE during 4 years follow-up, showed non-significant variations in methylation status of LINE-1. Conclusion Our study demonstrated the feasibility of our methodological approach to detect hypomethylation events in EADC patient cfDNA. Longitudinal preliminary analysis suggested a correlation between methylation status of LINE-1 and tumor progression. Further studies on larger population are needed to confirm these encouraging data. LINE-1 methylation analysis could be useful as a new molecular assay to integrate patient monitoring.

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Stearoyl-CoA Desaturase 1 Activity Determines the Maintenance of DNMT1-Mediated DNA Methylation Patterns in Pancreatic β-Cells

International Journal of Molecular Sciences ◽

10.3390/ijms21186844 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6844

Author(s):

Aneta M. Dobosz ◽

Justyna Janikiewicz ◽

Anna M. Borkowska ◽

Anna Dziewulska ◽

Ewelina Lipiec ◽

...

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Methylation Status ◽

Sirtuin 1 ◽

Dna Hypomethylation ◽

Β Cells ◽

Pancreatic Β Cells ◽

Compound C ◽

Stearoyl Coa Desaturase ◽

Methylation Patterns

Metabolic stress, such as lipotoxicity, affects the DNA methylation profile in pancreatic β-cells and thus contributes to β-cell failure and the progression of type 2 diabetes (T2D). Stearoyl-CoA desaturase 1 (SCD1) is a rate-limiting enzyme that is involved in monounsaturated fatty acid synthesis, which protects pancreatic β-cells against lipotoxicity. The present study found that SCD1 is also required for the establishment and maintenance of DNA methylation patterns in β-cells. We showed that SCD1 inhibition/deficiency caused DNA hypomethylation and changed the methyl group distribution within chromosomes in β-cells. Lower levels of DNA methylation in SCD1-deficient β-cells were followed by lower levels of DNA methyltransferase 1 (DNMT1). We also found that the downregulation of SCD1 in pancreatic β-cells led to the activation of adenosine monophosphate-activated protein kinase (AMPK) and an increase in the activity of the NAD-dependent deacetylase sirtuin-1 (SIRT1). Furthermore, the physical association between DNMT1 and SIRT1 stimulated the deacetylation of DNMT1 under conditions of SCD1 inhibition/downregulation, suggesting a mechanism by which SCD1 exerts control over DNMT1. We also found that SCD1-deficient β-cells that were treated with compound c, an inhibitor of AMPK, were characterized by higher levels of both global DNA methylation and DNMT1 protein expression compared with untreated cells. Therefore, we found that activation of the AMPK/SIRT1 signaling pathway mediates the effect of SCD1 inhibition/deficiency on DNA methylation status in pancreatic β-cells. Altogether, these findings suggest that SCD1 is a gatekeeper that protects β-cells against the lipid-derived loss of DNA methylation and provide mechanistic insights into the mechanism by which SCD1 regulates DNA methylation patterns in β-cells and T2D-relevant tissues.

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Epigenetic Contribution and Genomic Imprinting Dlk1-Dio3 miRNAs in Systemic Lupus Erythematosus

Genes ◽

10.3390/genes12050680 ◽

2021 ◽

Vol 12 (5) ◽

pp. 680

Author(s):

Rujuan Dai ◽

Zhuang Wang ◽

S. Ansar Ahmed

Keyword(s):

Systemic Lupus Erythematosus ◽

Dna Methylation ◽

Lupus Erythematosus ◽

Critical Role ◽

Methylation Status ◽

Transcriptional Level ◽

Dna Hypomethylation ◽

Systemic Lupus ◽

Multiple Organs ◽

Genetic Imprinting

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease that afflicts multiple organs, especially kidneys and joints. In addition to genetic predisposition, it is now evident that DNA methylation and microRNAs (miRNAs), the two major epigenetic modifications, are critically involved in the pathogenesis of SLE. DNA methylation regulates promoter accessibility and gene expression at the transcriptional level by adding a methyl group to 5′ cytosine within a CpG dinucleotide. Extensive evidence now supports the importance of DNA hypomethylation in SLE etiology. miRNAs are small, non-protein coding RNAs that play a critical role in the regulation of genome expression. Various studies have identified the signature lupus-related miRNAs and their functional contribution to lupus incidence and progression. In this review, the mutual interaction between DNA methylation and miRNAs regulation in SLE is discussed. Some lupus-associated miRNAs regulate DNA methylation status by targeting the DNA methylation enzymes or methylation pathway-related proteins. On the other hand, DNA hyper- and hypo-methylation are linked with dysregulated miRNAs expression in lupus. Further, we specifically discuss the genetic imprinting Dlk1-Dio3 miRNAs that are subjected to DNA methylation regulation and are dysregulated in several autoimmune diseases, including SLE.

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Abstract 40: Disturbed Flow Alters Genomewide DNA Methylation Patterns, Regulating Endothelial Gene Expression and Atherosclerosis

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.34.suppl_1.40 ◽

2014 ◽

Vol 34 (suppl_1) ◽

Author(s):

Jessilyn Dunn ◽

Haiwei Qiu ◽

Soyeon Kim ◽

Daudi Jjingo ◽

Ryan Hoffman ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Dna Methyltransferase ◽

Methylation Status ◽

Western Diet ◽

Dependent Manner ◽

Gene Promoters ◽

Genome Wide ◽

Methylation Patterns ◽

Endothelial Gene Expression

Atherosclerosis preferentially occurs in arterial regions of disturbed blood flow (d-flow), which alters gene expression, endothelial function, and atherosclerosis. Here, we show that d-flow regulates genome-wide DNA methylation patterns in a DNA methyltransferase (DNMT)-dependent manner. We found that d-flow induced expression of DNMT1, but not DNMT3a or DNMT3b, in mouse arterial endothelium in vivo and in cultured endothelial cells by oscillatory shear (OS) compared to unidirectional laminar shear in vitro. The DNMT inhibitor 5-Aza-2’deoxycytidine (5Aza) or DNMT1 siRNA significantly reduced OS-induced endothelial inflammation. Moreover, 5Aza reduced lesion formation in two atherosclerosis models using ApoE-/- mice (western diet for 3 months and the partial carotid ligation model with western diet for 3 weeks). To identify the 5Aza mechanisms, we conducted two genome-wide studies: reduced representation bisulfite sequencing (RRBS) and transcript microarray using endothelial-enriched gDNA and RNA, respectively, obtained from the partially-ligated left common carotid artery (LCA exposed to d-flow) and the right contralateral control (RCA exposed to s-flow) of mice treated with 5Aza or vehicle. D-flow induced DNA hypermethylation in 421 gene promoters, which was significantly prevented by 5Aza in 335 genes. Systems biological analyses using the RRBS and the transcriptome data revealed 11 mechanosensitive genes whose promoters were hypermethylated by d-flow but rescued by 5Aza treatment. Of those, five genes contain hypermethylated cAMP-response-elements in their promoters, including the transcription factors HoxA5 and Klf3. Their methylation status could serve as a mechanosensitive master switch in endothelial gene expression. Our results demonstrate that d-flow controls epigenomic DNA methylation patterns in a DNMT-dependent manner, which in turn alters endothelial gene expression and induces atherosclerosis.

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DNA methylation pattern during the encystment of Physarum flavicomum

Biochemistry and Cell Biology ◽

10.1139/o90-139 ◽

1990 ◽

Vol 68 (6) ◽

pp. 944-948 ◽

Cited By ~ 10

Author(s):

Chengming Zhu ◽

Henry R. Henney Jr.

Keyword(s):

Dna Methylation ◽

High Performance ◽

Restriction Enzymes ◽

Methylation Pattern ◽

Dna Base Composition ◽

Metabolic Role ◽

Growing Cell ◽

Dna Base ◽

Intracellular Content ◽

Methylation Patterns

In Physarum flavicomum Berk., haploid myxamoebae convert to dormant microcysts under conditions of nutrient imbalance. Exogenous adenine increases the intracellular content of S-adenosylmethionine (SAM) and inhibits this process. However, treatments that reduce the intracellular SAM levels relieve the inhibition of encystment induced by adenine. SAM plays a major metabolic role in cellular transmethylation reactions. In this study, we compared the DNA methylation patterns of growing cells, encysting cells, adenine-inhibited cells, and cysts using three different approaches: incubation of the cells with [14C]methylmethionine and detection of the labeled methyl group in purified DNA samples; analyses of DNA base composition by high performance liquid chromatography; and restriction endonuclease analyses of DNA. We found that DNA from the adenine-treated cells was labelled 1.3 times more with [14C]methylmethionine than was the DNA of untreated encysting cells. The DNA G + C content of this species was about 41%. The DNA of growing cells had the highest 5-methylcytosine (5MC) content, while DNA from the cysts had the lowest (about 27% that of growing cells). Adenine-inhibited cells had about 1.2 times more DNA-5MC than did encysting cells. Using the restriction enzymes SmaI, PvuI, and XhoI (which are inhibited by C residue methylation), we found that cyst DNA had more cutting sites than did amoebal DNA. By using the restriction enzyme DpnI which cuts DNA at GmATC sites, we found that cyst DNA, but not growing cell DNA, contained N6-methyladenine.Key words: amoebae, cysts, methylation, 5-methylcytosine, N6-methyladenine, DNA, encystment, Physarum flavicomum, development, inhibition.

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Methylation Profile of B-Chronic Lymphocytic Leukemia Cells.

Blood ◽

10.1182/blood.v106.11.2951.2951 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2951-2951

Author(s):

Jun Fan ◽

Asou Norio ◽

Masao Matsuoka

Keyword(s):

Dna Methylation ◽

Chronic Lymphocytic Leukemia ◽

Cell Lines ◽

Mammalian Cells ◽

Mononuclear Cells ◽

Cpg Island ◽

Methylation Status ◽

Lymphocytic Leukemia ◽

Dna Hypomethylation ◽

Peripheral Blood Mononuclear

Abstract DNA methylation plays an important role in the development and aging of mammalian cells, and its dysregulation has been frequently observed in cancer cells. The purpose of this study is to investigate the involvement of aberrant DNA methylation in B chronic lymphocytic leukemia (B-CLL) cells. We compared methylation status of B-CLL cells isolated from patients with that of normal CD19+ cells isolated from health donors by methylated CpG island amplification/representative difference analysis method. 5 hypermethylated and 27 hypomethylated DNA regions were identified in B-CLL sample. Among the 27 hypomethylated regions, 5 located on chromosome 9q34, 3 on 10q25-26 and 4 on 19q13. Methylation status was confirmed by sequencing using sodium bisulfite-treated DNA samples. By comparing DNA samples from same patients at different clinical stages, we found that lower methylation density in these regions is linked with disease progression. Expression of 15 genes surrounding hypomethylated regions was studied by RT-PCR. Expression of laminin beta3 gene and melanotransferrin gene was found to be upregulated in all B-CLL cell lines as well as lymphoma cell lines comparing with normal CD19+ peripheral blood mononuclear cells. B-cell CLL/lymphoma 11b gene showed increased expression in only 2 B-CLL cell lines. For other genes, no transcriptional change was found regardless of changed DNA methylation. This study showed the predominance of DNA hypomethylation in B-CLL cells compared with hypermethylation. Hypomethylated regions clustered in a limited number of chromosomes and methylation density appeared to be inversely correlated with disease progress. Figure Figure

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High Throughput Methylation Arrays Allow for Identification of Complex Methylation Patterns in MDS.

Blood ◽

10.1182/blood.v110.11.2437.2437 ◽

2007 ◽

Vol 110 (11) ◽

pp. 2437-2437

Author(s):

Ying Jiang ◽

Christine L. OKeefe ◽

Andrew Dunbar ◽

Anjali Advani ◽

Mikkael A. Sekeres ◽

...

Keyword(s):

Dna Methylation ◽

Tumor Suppressor ◽

High Throughput ◽

Tumor Suppressor Genes ◽

Methylation Status ◽

Epigenetic Silencing ◽

Low Grade ◽

High Grade ◽

Methylation Patterns ◽

Hypermethylated Genes

Abstract Genomic imprinting and epigenetic silencing determine tissue-specific methylation patterns. Altered methylation of CpG islands within gene promoters has been hypothesized as one pathogenetic mechanism operative in myelodysplastic syndrome (MDS). Promoter hypermethylation of various empirically selected tumor suppressor genes has been found in MDS prompting application of hypomethylating drugs in this disease. Identification of hypermethylated genes predicting response to these drugs would have a major impact on clinical practice. However, to date methylation-based prognostic algorithms have not been established. Global analysis of DNA methylation patterns may help to identify hypermethylated genes/promoters associated with the pathogenesis of MDS. Recently, microarray-based DNA methylation analysis platforms enabled a powerful, high-throughput analysis of the methylation status of hundreds of genes. The GoldenGate Methylation Cancer Panel I, spanning 1,536 independent CpG sites selected from 807 selected genes was applied to determine the methylation status in MDS patients (N=51; 21 low grade (RA, MDS-U, RARS or RCMD), 26 high grade (AML or RAEB) and 4 CMML). The methylation status was determined based on an internal reference and compared to healthy controls (N=22). Methylation values were averaged among the patients or analyzed separately for each patient in comparison to average values obtained in controls. Overall, controls showed a lesser degree of methylation than advanced MDS patients (average intensity 0.326 vs. 0.339, p<0.05). Subsequently, we concentrated on hypermethylated genes. There were no genes uniformly hypermethylated in all patients. For 70%, 50%, and 30% of patients with advanced MDS, 1, 26, and 85 loci were concordantly hypermethylated, while in 70%, 50% and 30% of low risk patients 5, 23 and 31 were hypermethylated, respectively. The most consistently hypermethylated genes (>50% of patients), included tumor suppressor genes (DCC, SLC22A18, FAT, TUSC3), genes involved in DNA repair (OGG1, DDB2, BCR, PARP1), cell cycle control (DBC1, SMARCB1), differentiation (MYOD1, TDGF1, FGF2, NOTCH4) and apoptosis (HDAC1, ALOX12, AXIN1). Despite the variability, the aberrant methylation spectrum in CMML, low grade MDS and high grade MDS showed significant overlap (for example FZD9, IL16, EVI2A, MBD2 and BCR), which suggests that these genes may relate to the common tumorigenesis in MDS. Certain genes show specific methylation correlating to the morphologic diagnosis and may serve as diagnostic markers. For example, the promoter of HDAC1 is hypomethylated in 81% of sAML/RAEB1/2 patients but hypermethylated in 81% of low risk cases. To assess the link between epigenetic changes and chromosomal abnormalities, we also investigated methylation pattern of MDS with del5q for selected genes at the 5q locus. Some genes that are involved in apoptosis (WNT1, TNF receptor) and proliferation (MAP3K8, CSF3) were found to be hypermethylated in comparison to controls, suggesting that epigenetic silencing may enhance the effect of haploinsuffciency for some of the genes. In sum, our study, the first application of a high-throughput microarray methylation assay in MDS, demonstrates that complex methylation patterns exist in MDS and may allow for identification for clinically relevant methylation markers.

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Characterization of DNA methylation of the promoters CTCF and BORIS (CTCFL) in breast and ovarian cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2013.31.15_suppl.e22151 ◽

2013 ◽

Vol 31 (15_suppl) ◽

pp. e22151-e22151 ◽

Cited By ~ 1

Author(s):

Ernesto Soto Reyes Solis ◽

Daniela Morales-Espinosa ◽

David Cantu ◽

Gabriela Alvarado-luna ◽

Dan Green ◽

...

Keyword(s):

Breast Cancer ◽

Ovarian Cancer ◽

Dna Methylation ◽

Methylation Status ◽

Dna Hypomethylation ◽

Rt Pcr ◽

Breast And Ovarian Cancer ◽

Neoplastic Tissue ◽

Tissue Samples ◽

Over Expression

e22151 Background: Genetic and epigenetic alterations may promote the initiation or development of cancer. Global DNA hypomethylation and local hypermethylation have been observed, particularly in cell cycle control-associated genes, such as tumor suppressor genes like CTCF. The dissociation of CTCF is associated with hypermethylation of several promoters; its paralogue gene (BORIS) is normally expressed in testicular tissue during spermatogenesis. BORIS over-expression has been identified in multiple neoplasms such as melanoma, gynecological cancer, glioblastoma and – recently – breast cancer. The aim of this study was to characterize the methylation status of the promoter regions of CTCF and BORIS in samples from breast and ovarian cancer compared to non-neoplastic tissue, and correlate it to its expression. Methods: Tissue samples from breast and ovarian cancer, as well as healthy controls were analyzed by MS-PCR for CTCF and BORIS. BorismRNA expression was also analyzed by RT-PCR. Results: A total of 8 ovarian and 16 breast tumors, as well as 10 tumor-adjacent breast tissue samples were prospectively obtained. In non-neoplastic tissue, BORIS was found to be hypermethylated, while in ovarian tumors a loss of methylation was identified in 75% of the samples. The same phenomenon was observed in 68% of breast cancer samples when compared to non-neoplastic tissue. A correlation between loss of DNA methylation of the promoter and gene over-expression was found by RT-PCR, thus suggesting that methylation is an epigenetic phenomenon associated to the over-expression of the oncogene BORIS. The methylation analysis of CTCF did not show any differences between neoplastic and non-neoplastic tissue, suggesting that epigenetic changes mainly affect BORIS. Conclusions: Loss of methylation of the promoter region of BORIS is associated with the over-expression of the gene. No differences were found in the methylation status between healthy and neoplastic tissue for CTCF.

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DNA methylation patterns predict clinical outcome in esophageal adenocarcinoma

Gastroenterology ◽

10.1016/s0016-5085(08)80150-3 ◽

2001 ◽

Vol 120 (5) ◽

pp. A31

Author(s):

Shahjehan A. Wajed ◽

Tiffany I. Long ◽

Cindy A. Eads ◽

Kumari Wickramasinghe ◽

Reginald V. Lord ◽

...

Keyword(s):

Dna Methylation ◽

Clinical Outcome ◽

Esophageal Adenocarcinoma ◽

Methylation Patterns

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Uhrf1 governs the proliferation and differentiation of muscle satellite cells

10.1101/2021.04.08.439096 ◽

2021 ◽

Author(s):

Hiroshi Sakai ◽

Yuichiro Sawada ◽

Naohito Tokunaga ◽

So Nakagawa ◽

Iori Sakakibara ◽

...

Keyword(s):

Dna Methylation ◽

Notch Signaling ◽

Satellite Cells ◽

Ring Finger ◽

Dna Hypomethylation ◽

Myogenic Cells ◽

Muscle Stem Cells ◽

Proliferation And Differentiation ◽

Essential Form ◽

Methylation Patterns

DNA methylation is an essential form of epigenetic regulation responsible for cellular identity. In muscle stem cells, termed satellite cells, DNA methylation patterns are tightly regulated during differentiation. However, it is unclear how these DNA methylation patterns are maintained. We demonstrate that a key epigenetic regulator, ubiquitin like with PHD and RING finger domains 1 (Uhrf1), is activated in proliferating myogenic cells but not expressed in quiescent or differentiated myogenic cells in mice. Ablation of Uhrf1 in mouse satellite cells impairs their proliferation and differentiation, leading to failed muscle regeneration. Loss of Uhrf1 in satellite cells alters transcriptional programs, leading to DNA hypomethylation with activation of Cdkn1a and Notch signaling. Although down-regulation of Cdkn1a rescued proliferation but not differentiation, inhibition of Notch signaling rescued impaired differentiation of Uhrf1-deficient satellite cells. These findings point to Uhrf1 as a regulator of self-renewal and differentiation of satellite cells via genome-wide DNA methylation patterning.

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