scholarly journals Locus-Specific DNA Methylation Editing in Melanoma Cell Lines Using a CRISPR-Based System

Cancers ◽  
2021 ◽  
Vol 13 (21) ◽  
pp. 5433
Author(s):  
Jim Smith ◽  
Rakesh Banerjee ◽  
Reema Waly ◽  
Arthur Urbano ◽  
Gregory Gimenez ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Epigenetic Modification ◽  
Gene Promoter ◽  
Human Melanoma ◽  
Melanoma Metastasis ◽  
Guide Rna ◽  
Targeted Screening ◽  
Transcriptional Deregulation ◽  
Transient System ◽  
Target Activity

DNA methylation is a key epigenetic modification implicated in the pathogenesis of numerous human diseases, including cancer development and metastasis. Gene promoter methylation changes are widely associated with transcriptional deregulation and disease progression. The advent of CRISPR-based technologies has provided a powerful toolkit for locus-specific manipulation of the epigenome. Here, we describe a comprehensive global workflow for the design and application of a dCas9-SunTag-based tool for editing the DNA methylation locus in human melanoma cells alongside protocols for downstream techniques used to evaluate subsequent methylation and gene expression changes in methylation-edited cells. Using transient system delivery, we demonstrate both highly efficacious methylation and demethylation of the EBF3 promoter, which is a putative epigenetic driver of melanoma metastasis, achieving up to a 304.00% gain of methylation and 99.99% relative demethylation, respectively. Furthermore, we employ a novel, targeted screening approach to confirm the minimal off-target activity and high on-target specificity of our designed guide RNA within our target locus.

scholarly journals Locus-Specific DNA Methylation Editing in Mammalian Cells using a CRISPR-Based System

2021 ◽  
Author(s):  
Jim Smith ◽  
Rakesh Banerjee ◽  
Reema Waly ◽  
Arthur Urbano ◽  
Gregory Gimenez ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Mammalian Cells ◽  
Epigenetic Modification ◽  
Gene Promoter ◽  
Human Melanoma ◽  
Melanoma Metastasis ◽  
Targeted Screening ◽  
Transcriptional Deregulation ◽  
Transient System ◽  
Target Activity

AbstractDNA methylation is a key epigenetic modification implicated in the pathogenesis of numerous human diseases, including cancer development and metastasis. Gene promoter methylation changes are widely associated with transcriptional deregulation and disease progression. The advent of CRISPR-based technologies has provided a powerful toolkit for locus-specific manipulation of the epigenome. Here, we describe a comprehensive global workflow for the design and application of a dCas9-SunTag-based tool for editing a DNA methylation locus in human melanoma cells, alongside protocols for downstream techniques used to evaluate subsequent methylation and gene expression changes in methylation-edited cells. Using transient system delivery, we demonstrate both highly efficacious methylation and demethylation of the EBF3 promoter, a putative epigenetic driver of melanoma metastasis, achieving up to 304.00% gain of methylation and 99.99% relative demethylation, respectively. Further, we employ a novel, targeted screening approach to confirm minimal off-target activity and high on-target specificity of our editing sys-tem within our target locus.

Catechol-O-methyltransferase gene promoter methylation as a peripheral biomarker in male schizophrenia

2017 ◽  
Vol 44 ◽  
pp. 39-46 ◽  
Author(s):  
S. Gao ◽  
J. Cheng ◽  
G. Li ◽  
T. Sun ◽  
Y. Xu ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Operating Characteristic ◽  
Epigenetic Modification ◽  
Gene Promoter ◽  
Clinical Value ◽  
Bisulfite Pyrosequencing ◽  
Methyltransferase Gene ◽  
Genetic And Environmental Factors ◽  
Male Specific ◽  
Pyrosequencing Technology

AbstractAs an epigenetic modification, DNA methylation may reflect the interaction between genetic and environmental factors in the development of schizophrenia (SCZ). Catechol-O-methyltransferase (COMT) gene is a promising candidate gene of SCZ. In the present study, we investigate the association of COMT methylation with the risk of SCZ using bisulfite pyrosequencing technology. Significant association between DNA methylation of COMT and the risk of SCZ is identified (P = 1.618e−007). A breakdown analysis by gender shows that the significance is driven by males (P = 3.310e−009), but not by females. DNA methylation of COMT is not significantly associated with SCZ clinical phenotypes, including p300 and cysteine level. No interaction is found between COMT genotypes and the percent methylation of this gene. Receiver operating characteristic (ROC) curve shows that DNA methylation of COMT is able to predict the SCZ risk in males (area under curve [AUC] = 0.802, P = 1.91e−007). The current study indicates the clinical value of COMT methylation as a potential male-specific biomarker in SCZ diagnosis.

Aberrant DNA Methylation Patterns in Chronic Idiopathic Myelofibrosis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2443-2443
Author(s):  
Edgar Jost ◽  
Gerald Gehbauer ◽  
Christoph E. Maintz ◽  
Peter Jousten ◽  
Leo Habets ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Tumor Suppressor ◽  
Tumor Suppressor Genes ◽  
Epigenetic Modification ◽  
Cpg Islands ◽  
Gene Promoter ◽  
Aberrant Methylation ◽  
Idiopathic Myelofibrosis ◽  
Suppressor Genes ◽  
Chronic Idiopathic Myelofibrosis

Abstract Chronic idiopathic myelofibrosis (CIMF) is a clonal myeloproliferative disorder characterized by bone marrow fibrosis, angiogenesis and extramedullary hematopoiesis. No specific genetic defect underlying the disease has been identified so far. The spectrum of cytogenetic abnormalities in CIMF includes del (13q), del (20q) and partial trisomy 1q as well as abnormalities of chromosomes 1, 7 and 9. Hypermethylation of CpG islands within gene promoter regions is associated with transcriptional inactivation and represents an important mechanism of gene silencing in the pathogenesis of hematopoietic malignancies. This epigenetic phenomenon acts as an alternative to mutations and deletions to disrupt tumor suppressor gene function in cancerogenesis. In order to investigate the role of DNA methylation changes in the pathogenesis of CIMF, we have analyzed the methylation status of the promoter-associated CpG islands of 13 well-characterized tumor suppressor genes by methylation-specific polymerase chain reaction in peripheral blood cells from 20 adult patients with CIMF. The frequency of aberrant methylation among the patient samples was 25.0 % (5/20) for SOCS-1 and 5.0 % (1/20) for E-cadherin, MGMT, p73 as well as TIMP-2. There was no hypermethylation of p15, p16, RARbeta, DAP kinase 1, SOCS-3, hMLH1, TIMP-3 and RASSF1A. We detected at least one hypermethylated gene promoter region in 35.0 % (7/20) of the primary patient samples. Our data indicate that hypermethylation of tumor suppressor genes is a common event in CIMF. Epigenetic modification of genes regulating growth factor signaling, cell adhesion and DNA repair may, in addition to genetic aberrations, contribute to the malignant phenotype in CIMF. The exploration of our growing knowledge about epigenetic aberrations in tumorigenesis may help develop novel strategies in diagnosis and treatment of CIMF for the future.

scholarly journals Expression of Sclerostin in Osteoporotic Fracture Patients Is Associated with DNA Methylation in the CpG Island of the SOST Gene

2019 ◽  
Vol 2019 ◽  
pp. 1-8 ◽  
Author(s):  
Yanming Cao ◽  
Bin Wang ◽  
Ding Wang ◽  
Dongxiang Zhan ◽  
Caiyuan Mai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Osteoporotic Fracture ◽  
Cpg Island ◽  
Epigenetic Modification ◽  
Methylation Status ◽  
Gene Promoter ◽  
Primary Osteoporosis ◽  
Sost Gene ◽  
Fracture Bone

Purpose. SOST gene is one of the key factors in regulating bone absorption. Although there are reports showing diverse transcription factors, epigenetic modification could be responsible for regulating SOST gene expression. There is still little exploration on promoter methylation status of SOST gene in osteoporotic bone tissues. The aim of this study is to investigate the involvement of CpG methylation in regulation of SOST expression in patients with primary osteoporosis. Methods. The diagnosis of osteoporosis was established on the basis of dual energy X-ray absorptiometry to measure BMD. All femoral bone tissues were separated in surgeries. After extracting total RNA and protein, we checked the relative expression levels of SOST by quantitative real-time PCR and western blot. Also, immunohistochemical staining was performed to observe the expression of SOST protein in the bone samples. The genomic DNA of non-OPF (non-osteoporotic fracture bone tissues) and OPF (osteoporotic fracture bone tissues) were treated by bisulfite modification, and methylation status of CpG sites in the CpG island of SOST gene promoter was determined by DNA sequencing. Results. SOST gene expression in the non-OPF group was lower than that in OPF group. Bisulfite sequencing result showed that SOST gene promoter was slightly demethylated in the OPF group, as compared with non-OPF group. Conclusion. Our study demonstrated that DNA methylation influenced the transcriptional expression of SOST gene, which probably may play an important role in the pathogenesis of primary osteoporosis.

scholarly journals The Pattern and Function of DNA Methylation in Fungal Plant Pathogens

2020 ◽  
Vol 8 (2) ◽  
pp. 227 ◽  
Author(s):  
Chang He ◽  
Zhanquan Zhang ◽  
Boqiang Li ◽  
Shiping Tian
Keyword(s):  
Dna Methylation ◽  
Dna Sequences ◽  
Plant Pathogens ◽  
Epigenetic Modification ◽  
Gene Promoter ◽  
Pathogenic Fungi ◽  
Dna Methyltransferases ◽  
Phytopathogenic Fungi ◽  
Fungal Plant Pathogens ◽  
Wide Range

To successfully infect plants and trigger disease, fungal plant pathogens use various strategies that are dependent on characteristics of their biology and genomes. Although pathogenic fungi are different from animals and plants in the genomic heritability, sequence feature, and epigenetic modification, an increasing number of phytopathogenic fungi have been demonstrated to share DNA methyltransferases (MTases) responsible for DNA methylation with animals and plants. Fungal plant pathogens predominantly possess four types of DNA MTase homologs, including DIM-2, DNMT1, DNMT5, and RID. Numerous studies have indicated that DNA methylation in phytopathogenic fungi mainly distributes in transposable elements (TEs), gene promoter regions, and the repetitive DNA sequences. As an important and heritable epigenetic modification, DNA methylation is associated with silencing of gene expression and transposon, and it is responsible for a wide range of biological phenomena in fungi. This review highlights the relevant reports and insights into the important roles of DNA methylation in the modulation of development, pathogenicity, and secondary metabolism of fungal plant pathogens. Recent evidences prove that there are massive links between DNA and histone methylation in fungi, and they commonly regulate fungal development and mycotoxin biosynthesis.

scholarly journals Unraveling the functional role of DNA demethylation at specific promoters by targeted steric blockage of DNA methyltransferase with CRISPR/dCas9

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Daniel M. Sapozhnikov ◽  
Moshe Szyf
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Living Cells ◽  
Dna Demethylation ◽  
Inducible Promoters ◽  
Guide Rna ◽  
Per Se ◽  
Target Activity

AbstractDespite four decades of research to support the association between DNA methylation and gene expression, the causality of this relationship remains unresolved. Here, we reaffirm that experimental confounds preclude resolution of this question with existing strategies, including recently developed CRISPR/dCas9 and TET-based epigenetic editors. Instead, we demonstrate a highly effective method using only nuclease-dead Cas9 and guide RNA to physically block DNA methylation at specific targets in the absence of a confounding flexibly-tethered enzyme, thereby enabling the examination of the role of DNA demethylation per se in living cells, with no evidence of off-target activity. Using this method, we probe a small number of inducible promoters and find the effect of DNA demethylation to be small, while demethylation of CpG-rich FMR1 produces larger changes in gene expression. This method could be used to reveal the extent and nature of the contribution of DNA methylation to gene regulation.

EPIGENETIC ALTERATIONS ASSOCIATED WITH PREMATURE OVARIAN FAILURE

2008 ◽  
Vol 31 (4) ◽  
pp. 11
Author(s):  
Manda Ghahremani ◽  
Courtney W Hannah ◽  
Maria Peneherrera ◽  
Karla L Bretherick ◽  
Margo R Fluker ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Premature Ovarian Failure ◽  
Promoter Region ◽  
Gene Promoter ◽  
Ovarian Failure ◽  
P Value ◽  
Epigenetic Alterations ◽  
Methylation Array ◽  
Cpg Sites ◽  
Deficient Mice

Background/Purpose: Premature ovarian failure (POF) affects 1% of women with a largely idiopathic and poorly understood etiology. The objective of this study was to identify specific epigenetic alterations by measuring DNA methylation of gene regulatory regions in women with POF vs. controls. Methods: Blood samples were collected from idiopathic POFpatients (Amenorrhea for at least 3 months and 2 serum FSH levels of > 40mIU/ml obtained > 1 month apart prior to age 40) and control women (CW) (healthy pregnancy after age 37 with out a pregnancy loss). Genomic DNA was extracted from EDTA anticoagulated blood and bisulfite converted for analysis using the Illumina Golden Gate Methylation Panel which measures DNA methylation at 1506 CpG sites in the promoter regions of 807 genes in 10 POF and 12 CW. Candidate genes with altered epigenetic marks between POF and CW at a nominal P-value < 0.05 were identified using a t-testcomparison within the Illumina bead studio software. Genes of interest were further analyzed for quantitative methylation at specific CpG sites using pyrosequencing in 30 POF and 30 CW. Results: Comparison of DNA methylation profiles of our initial POF and CW groups identified several genes with statistically significanthyper- or hypo- methylation in the POF group (P < 0.05), including the Androgen Receptor (AR)promoter region, which was significantly hypermethylated. To further validate these results, DNA methylation of the AR gene promoter was quantified bypryosequencing in a larger group of POF and CW. Pyrosequencing further confirmed a significantly higher DNA methylation of the AR promoter region inPOF vs. CW (P=0.007). Conclusions: This is a novel study identifying epigenetic alterations in POF. The hypermethylation of the AR gene in POF patients may cause decreased level of the AR in these women. This is especially interesting given a recent report of induced POF in AR deficient mice^1. Specific epigenetic markers, as identified by DNA methylation array profiling in blood, may serve as useful biomarkers for POF and other fertility disorders. However, it will need to be determined if these methylation changes are present prior to diagnosis, or are a consequence of menopause itself. Reference: 1.Hiroko S. et al. Premature ovarian failure in androgenreceptor deficient mice. PNAS;103:224-9

Examination of the dynamics of global DNA methylation pattern establishment during spermatogenesiss

2007 ◽  
Vol 30 (4) ◽  
pp. 90
Author(s):  
Kirsten Niles ◽  
Sophie La Salle ◽  
Christopher Oakes ◽  
Jacquetta Trasler
Keyword(s):  
Dna Methylation ◽  
Germ Cell ◽  
Germ Cells ◽  
Epigenetic Modification ◽  
Methylation Pattern ◽  
Chromosome 9 ◽  
Specific Gene ◽  
Global Methylation ◽  
Dna Methylation Pattern ◽  
Methylation Patterns

Background: DNA methylation is an epigenetic modification involved in gene expression, genome stability, and genomic imprinting. In the male, methylation patterns are initially erased in primordial germ cells (PGCs) as they enter the gonadal ridge; methylation patterns are then acquired on CpG dinucleotides during gametogenesis. Correct pattern establishment is essential for normal spermatogenesis. To date, the characterization and timing of methylation pattern acquisition in PGCs has been described using a limited number of specific gene loci. This study aimed to describe DNA methylation pattern establishment dynamics during male gametogenesis through global methylation profiling techniques in a mouse model. Methods: Using a chromosome based approach, primers were designed for 24 regions spanning chromosome 9; intergenic, non-repeat, non-CpG island sequences were chosen for study based on previous evidence that these types of sequences are targets for testis-specific methylation events. The percent methylation was determined in each region by quantitative analysis of DNA methylation using real-time PCR (qAMP). The germ cell-specific pattern was determined by comparing methylation between spermatozoa and liver. To examine methylation in developing germ cells, spermatogonia from 2 day- and 6 day-old Oct4-GFP (green fluorescent protein) mice were isolated using fluorescence activated cell sorting. Results: As compared to liver, four loci were hypomethylated and five loci were hypermethylated in spermatozoa, supporting previous results indicating a unique methylation pattern in male germ cells. Only one region was hypomethylated and no regions were hypermethylated in day 6 spermatogonia as compared to mature spermatozoa, signifying that the bulk of DNA methylation is established prior to type A spermatogonia. The methylation in day 2 spermatogonia, germ cells that are just commencing mitosis, revealed differences of 15-20% compared to day 6 spermatogonia at five regions indicating that the most crucial phase of DNA methylation acquisition occurs prenatally. Conclusion: Together, these studies provide further evidence that germ cell methylation patterns differ from those in somatic tissues and suggest that much of methylation at intergenic sites is acquired during prenatal germ cell development. (Supported by CIHR)

ASSESSMENT OF EFFICIENCY AND OFF-TARGET ACTIVITY OF CRISPR/CAS RIBONUCLEOPROTEIN COMPLEXES

2020 ◽  
Author(s):  
Y.V. Mikhaylova ◽  
◽  
M.A. Tyumentseva ◽  
A.A. Shelenkov ◽  
Y.G. Yanushevich ◽  
...  
Keyword(s):  
High Sensitivity ◽  
Correct Choice ◽  
Target Sequence ◽  
Dna Breaks ◽  
Gene Encoding ◽  
Guide Rna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Chemokine Receptor Ccr5 ◽  
Target Activity

In this study, we assessed the efficiency and off-target activity of the CRISPR/CAS complex with one of the selected guide RNAs using the CIRCLE-seq technology. The gene encoding the human chemokine receptor CCR5 was used as a target sequence for genome editing. The results of this experiment indicate the correct choice of the guide RNA and efficient work of the CRISPR- CAS ribonucleoprotein complex used. CIRCLE-seq technology has shown high sensitivity compared to bioinformatic methods for predicting off-target activity of CRISPR/CAS complexes. We plan to evaluate the efficiency and off-target activity of CRISPR/CAS ribonucleoprotein complexes with other guide RNAs by slightly adjusting the CIRCLE-seq-technology protocol in order to reduce nonspecific DNA breaks and increase the number of reliable reads.

Epigenetic modifications in acute lymphoblastic leukemia: From cellular mechanisms to therapeutics

2020 ◽  
Vol 20 ◽  
Author(s):  
Ezzatollah Fathi ◽  
Raheleh Farahzadi ◽  
Soheila Montazersaheb ◽  
Yasin Bagheri
Keyword(s):  
Dna Methylation ◽  
Acute Lymphoblastic Leukemia ◽  
Histone Modification ◽  
Epigenetic Modification ◽  
Lymphoblastic Leukemia ◽  
Underlying Mechanism ◽  
Methylation Pattern ◽  
Epigenetic Alterations ◽  
Cellular Mechanisms ◽  
Novel Treatments

Background:: Epigenetic modification pattern is considered as a characteristic feature in blood malignancies. Modifications in the DNA methylation modulators are recurrent in lymphoma and leukemia, so that, the distinct methylation pattern defines different types of leukemia. Generally, the role of epigenetics is less understood and most investigations are focused on genetic abnormalities and cytogenic studies to develop novel treatments for patients with hematologic disorders. Recently, understanding the underlying mechanism of acute lymphoblastic leukemia (ALL), especially epigenetic altera-tions as a driving force in the development of ALL opens a new era of investigation for developing promising strategy, be-yond available conventional therapy. Objective:: This review will focus on a better understanding of the epigenetic mechanisms in cancer development and pro-gression, with an emphasis on epigenetic alterations in ALL including, DNA methylation, histone modification, and mi-croRNA alterations. Other topics that will be discussed include the use of epigenetic alterations as a promising therapeutic target in order to develop novel well-suited approaches against ALL. Conclusion:: According to the literature review, leukemogenesis of ALL is extensively influenced by epigenetic modifica-tions, particularly DNA hyper-methylation, histone modification, and miRNA alteration.

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