Conserved Methylation Patterns of Human Papillomavirus Type 16 DNA in Asymptomatic Infection and Cervical Neoplasia

ABSTRACT DNA methylation contributes to the chromatin conformation that represses transcription of human papillomavirus type16 (HPV-16), which is prevalent in the etiology of cervical carcinoma. In an effort to clarify the role of this phenomenon in the regulation and carcinogenicity of HPV-16, 115 clinical samples were studied to establish the methylation patterns of the 19 CpG dinucleotides within the long control region and part of the L1 gene by bisulfite modification, PCR amplification, DNA cloning, and sequencing. We observed major heterogeneities between clones from different samples as well as between clones from individual samples. The methylation frequency of CpGs was measured at 14.5%. In addition, 0.21 and 0.23%, respectively, of the CpA and CpT sites, indicators of de novo methylation, were methylated. Methylation frequencies exceeded 30% in the CpGs overlapping with the L1 gene and were about 10% for most other positions. A CpG site located in the linker between two nucleosomes positioned over the enhancer and promoter of HPV-16 had minimal methylation. This region forms part of the HPV replication origin and is close to binding sites of master-regulators of transcription during epithelial differentiation. Methylation of most sites was highest in carcinomas, possibly due to tandem repetition and chromosomal integration of HPV-16 DNA. Methylation was lowest in dysplasia, likely reflecting the transcriptional activity in these infections. Our data document the efficient targeting of HPV genomes by the epithelial methylation machinery, possibly as a cellular defense mechanism, and suggest involvement of methylation in HPV oncogene expression and the early-late switch.

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DNA Methylation Changes in Human Papillomavirus-Driven Head and Neck Cancers

Cells ◽

10.3390/cells9061359 ◽

2020 ◽

Vol 9 (6) ◽

pp. 1359 ◽

Cited By ~ 1

Author(s):

Chameera Ekanayake Weeramange ◽

Kai Dun Tang ◽

Sarju Vasani ◽

Julian Langton-Lockton ◽

Liz Kenny ◽

...

Keyword(s):

Dna Methylation ◽

Human Papillomavirus ◽

Head And Neck ◽

Dna Methyltransferase ◽

Long Control Region ◽

Cancer Types ◽

E6 And E7 ◽

Hpv Oncoproteins ◽

Cellular Dna ◽

Methylation Patterns

Disruption of DNA methylation patterns is one of the hallmarks of cancer. Similar to other cancer types, human papillomavirus (HPV)-driven head and neck cancer (HNC) also reveals alterations in its methylation profile. The intrinsic ability of HPV oncoproteins E6 and E7 to interfere with DNA methyltransferase activity contributes to these methylation changes. There are many genes that have been reported to be differentially methylated in HPV-driven HNC. Some of these genes are involved in major cellular pathways, indicating that DNA methylation, at least in certain instances, may contribute to the development and progression of HPV-driven HNC. Furthermore, the HPV genome itself becomes a target of the cellular DNA methylation machinery. Some of these methylation changes appearing in the viral long control region (LCR) may contribute to uncontrolled oncoprotein expression, leading to carcinogenesis. Consistent with these observations, demethylation therapy appears to have significant effects on HPV-driven HNC. This review article comprehensively summarizes DNA methylation changes and their diagnostic and therapeutic indications in HPV-driven HNC.

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CpG methylation of an X-linked transgene is determined by somatic events postfertilization and not germline imprinting

Development ◽

10.1242/dev.104.2.235 ◽

1988 ◽

Vol 104 (2) ◽

pp. 235-244

Author(s):

A. Collick ◽

W. Reik ◽

S.C. Barton ◽

A.H. Surani

Keyword(s):

Dna Methylation ◽

X Chromosome ◽

De Novo ◽

Cpg Methylation ◽

Parental Origin ◽

Cpg Dinucleotides ◽

Inactive State ◽

Methylation Of Dna ◽

Methylation Patterns ◽

Extraembryonic Tissues

The process of X-inactivation in mammals requires at least two events, the initiation of inactivation and the maintenance of the inactive state. One possible mechanism of control is by methylation of DNA at CpG dinucleotides to maintain the inactive state. Furthermore, the paternal X-chromosome is frequently inactivated in the extraembryonic membranes. The relationship between the parental origin of the chromosome, nonrandom inactivation and DNA methylation is not clear. In this paper, we report on the CpG methylation of an X-linked transgene, CAT-32. The levels of methylation in embryonic, extraembryonic and germline cells indicates that the modifications of the transgene are broadly similar to those reported for endogenous X-linked genes. Interestingly, the methylation of CAT-32 transgene in extraembryonic tissues displays patterns that could be linked to the germline origin of each allele. Hence, the maternally derived copy of CAT-32 was relatively undermethylated when compared to the paternal one. The changes in DNA methylation were attributed to de novo methylation occurring after fertilization, most probably during differentiation of extraembryonic tissues. In order to determine whether or not the patterns of DNA methylation reflected the germline origin of the X-chromosome, we constructed triploid embryos specifically to introduce two maternal X-chromosomes in the same embryo. In some of these triploid conceptuses, methylation patterns characteristic of the paternally derived transgene were observed. This observation indicates that the methylation patterns are not necessarily dependent on the parental origin of the X-chromosome, but could be changed by somatic events after fertilization. One of the more likely mechanisms is methylation of the transgene following inactivation of the X-chromosome in extraembryonic tissues.

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Comparison of Variant-Specific Hybridization and Single-Strand Conformational Polymorphism Methods for Detection of Mixed Human Papillomavirus Type 16 Variant Infections

Journal of Clinical Microbiology ◽

10.1128/jcm.37.11.3627-3633.1999 ◽

1999 ◽

Vol 37 (11) ◽

pp. 3627-3633 ◽

Cited By ~ 10

Author(s):

Rebecca T. Emeny ◽

John R. Herron ◽

Long Fu Xi ◽

Laura A. Koutsky ◽

Nancy B. Kiviat ◽

...

Keyword(s):

Human Papillomavirus ◽

Single Strand ◽

Sscp Analysis ◽

Clinical Samples ◽

Long Control Region ◽

Single Strand Conformational Polymorphism ◽

Conformational Polymorphism ◽

Hpv 16 ◽

Human Papillomavirus Type 16 ◽

Specific Hybridization

PCR-based variant-specific hybridization (VSH) and single-strand conformational polymorphism (SSCP) analyses were compared for their capacities to detect mixed human papillomavirus type 16 (HPV-16) variant infections within clinical specimens. The SSCP assay used in this comparison targets a 682-bp fragment that spans nucleotides 7445 to 222 within the HPV-16 genome. This fragment includes portions of the HPV-16 long control region and the E6 open reading frame and identifies three categories of SSCP patterns: those identical to the patterns of prototype HPV-16 (P), those identical to the patterns of Caski-derived HPV-16 (C), or those that are different from the P and C HPV-16 patterns and that are therefore classified as belonging to novel (N) HPV-16 variants. VSH targets the entire HPV-16 E6-coding region (nucleotides 56 to 640) and distinguishes previously described variant nucleotides at positions 109, 131, 132, 143, 145, 178, 286, 289, 350, 403, and 532. Clinical samples used in VSH and SSCP analyses were subjected to multiple independent amplification reactions. The resultant amplicons were cloned, and 14 to 78 clones per clinical specimen were evaluated by VSH. VSH detected an HPV-16 variant that represented at least 20% of the amplified HPV-16 variant population. In contrast, SSCP analysis detected HPV-16 variants that represented 36% of the amplified HPV-16 population. Comparison studies were conducted with mixed HPV-16 variant laboratory constructs. Again, VSH had a higher sensitivity than SSCP analysis in detecting mixed HPV-16 variant infections in these constructed amplicon targets. Accurate detection of HPV-16 variants may enhance our understanding of the natural history of HPV-16 infections.

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Comparative analysis of DNA methylation patterns in transgenic Drosophila overexpressing mouse DNA methyltransferases

Biochemical Journal ◽

10.1042/bj20031567 ◽

2004 ◽

Vol 378 (3) ◽

pp. 763-768 ◽

Cited By ~ 32

Author(s):

Cora MUND ◽

Tanja MUSCH ◽

Martin STRÖDICKE ◽

Birte ASSMANN ◽

En LI ◽

...

Keyword(s):

Dna Methylation ◽

Mammalian Cells ◽

Cytosine Methylation ◽

De Novo ◽

Epigenetic Modification ◽

Dna Methyltransferases ◽

Significant Activity ◽

Cpg Dinucleotides ◽

Methylation Patterns ◽

Transgenic Drosophila

DNA methyltransferases (Dnmts) mediate the epigenetic modification of eukaryotic genomes. Mammalian DNA methylation patterns are established and maintained by co-operative interactions among the Dnmt proteins Dnmt1, Dnmt3a and Dnmt3b. Owing to their simultaneous presence in mammalian cells, the activities of individual Dnmt have not yet been determined. This includes a fourth putative Dnmt, namely Dnmt2, which has failed to reveal any activity in previous assays. We have now established transgenic Drosophila strains that allow for individual overexpression of all known mouse Dnmts. Quantitative analysis of genomic cytosine methylation levels demonstrated a robust Dnmt activity for the de novo methyltransferases Dnmt3a and Dnmt3b. In addition, we also detected a weak but significant activity for Dnmt2. Subsequent methylation tract analysis by genomic bisulphite sequencing revealed that Dnmt3 enzymes preferentially methylated CpG dinucleotides in a processive manner, whereas Dnmt2 methylated isolated cytosine residues in a non-CpG dinucleotide context. Our results allow a direct comparison of the activities of mammalian Dnmts and suggest a significant functional specialization of these enzymes.

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Structural insights into CpG-specific DNA methylation by human DNA methyltransferase 3B

Nucleic Acids Research ◽

10.1093/nar/gkaa111 ◽

2020 ◽

Vol 48 (7) ◽

pp. 3949-3961 ◽

Cited By ~ 5

Author(s):

Chien-Chu Lin ◽

Yi-Ping Chen ◽

Wei-Zen Yang ◽

James C K Shen ◽

Hanna S Yuan

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Cytosine Methylation ◽

Catalytic Domain ◽

De Novo ◽

Water Channel ◽

Dna Methyltransferases ◽

Structural Basis ◽

Cpg Sites ◽

Methylation Patterns

Abstract DNA methyltransferases are primary enzymes for cytosine methylation at CpG sites of epigenetic gene regulation in mammals. De novo methyltransferases DNMT3A and DNMT3B create DNA methylation patterns during development, but how they differentially implement genomic DNA methylation patterns is poorly understood. Here, we report crystal structures of the catalytic domain of human DNMT3B–3L complex, noncovalently bound with and without DNA of different sequences. Human DNMT3B uses two flexible loops to enclose DNA and employs its catalytic loop to flip out the cytosine base. As opposed to DNMT3A, DNMT3B specifically recognizes DNA with CpGpG sites via residues Asn779 and Lys777 in its more stable and well-ordered target recognition domain loop to facilitate processive methylation of tandemly repeated CpG sites. We also identify a proton wire water channel for the final deprotonation step, revealing the complete working mechanism for cytosine methylation by DNMT3B and providing the structural basis for DNMT3B mutation-induced hypomethylation in immunodeficiency, centromere instability and facial anomalies syndrome.

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Nurse cell-derived small RNAs define paternal epigenetic inheritance in Arabidopsis

10.1101/2021.01.25.428150 ◽

2021 ◽

Author(s):

Jincheng Long ◽

James Walker ◽

Wenjing She ◽

Billy Aldridge ◽

Hongbo Gao ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Small Rnas ◽

Nurse Cell ◽

De Novo ◽

Epigenetic Inheritance ◽

Genome Integrity ◽

Nurse Cells ◽

Male Germline ◽

Methylation Patterns

AbstractThe plant male germline undergoes DNA methylation reprogramming, which methylates genes de novo and thereby alters gene expression and facilitates meiosis. Why reprogramming is limited to the germline and how specific genes are chosen is unknown. Here, we demonstrate that genic methylation in the male germline, from meiocytes to sperm, is established by germline-specific siRNAs transcribed from transposons with imperfect sequence homology. These siRNAs are synthesized by meiocyte nurse cells (tapetum) via activity of the tapetum-specific chromatin remodeler CLASSY3. Remarkably, tapetal siRNAs govern germline methylation throughout the genome, including the inherited methylation patterns in sperm. Finally, we demonstrate that these nurse cell-derived siRNAs (niRNAs) silence germline transposons, thereby safeguarding genome integrity. Our results reveal that tapetal niRNAs are sufficient to reconstitute germline methylation patterns and drive extensive, functional methylation reprogramming analogous to piRNA-mediated reprogramming in animal germlines.

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Persistent epigenetic memory impedes rescue of the telomeric phenotype in human ICF iPSCs following DNMT3B correction

eLife ◽

10.7554/elife.47859 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 3

Author(s):

Shir Toubiana ◽

Miriam Gagliardi ◽

Mariarosaria Papa ◽

Roberta Manco ◽

Maty Tzukerman ◽

...

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

De Novo ◽

Pharmacological Intervention ◽

Icf Syndrome ◽

Genome Wide ◽

Mammalian Genomes ◽

Induced Pluripotent ◽

Methylation Patterns

DNA methyltransferase 3B (DNMT3B) is the major DNMT that methylates mammalian genomes during early development. Mutations in human DNMT3B disrupt genome-wide DNA methylation patterns and result in ICF syndrome type 1 (ICF1). To study whether normal DNA methylation patterns may be restored in ICF1 cells, we corrected DNMT3B mutations in induced pluripotent stem cells from ICF1 patients. Focusing on repetitive regions, we show that in contrast to pericentromeric repeats, which reacquire normal methylation, the majority of subtelomeres acquire only partial DNA methylation and, accordingly, the ICF1 telomeric phenotype persists. Subtelomeres resistant to de novo methylation were characterized by abnormally high H3K4 trimethylation (H3K4me3), and short-term reduction of H3K4me3 by pharmacological intervention partially restored subtelomeric DNA methylation. These findings demonstrate that the abnormal epigenetic landscape established in ICF1 cells restricts the recruitment of DNMT3B, and suggest that rescue of epigenetic diseases with genome-wide disruptions will demand further manipulation beyond mutation correction.

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DNA METHYLATION IN PLANTS

Journal of Global Innovations in Agriculture Sciences ◽

10.22194/jgias/9.9543 ◽

2021 ◽

pp. 109-114

Author(s):

Adil Altaf ◽

Ahmad Zada

Keyword(s):

Dna Methylation ◽

De Novo ◽

Epigenetic Inheritance ◽

Plant Tissues ◽

Developmental Abnormalities ◽

Process Use ◽

Model Plant Arabidopsis Thaliana ◽

Methylation Patterns ◽

Kinetics Of ◽

Generation Sequencing

Common DNA methylation controls gene expression and preserves genomic integrity. Mal methylation can cause developmental abnormalities in the plants. Multiple enzymes carrying out de novo methylation, methylation maintenance, and active demethylation culminate in a particular DNA methylation state. Next-generation sequencing advances and computational methods to analyze the data. The model plant Arabidopsis thaliana was used to study DNA methylation patterns, epigenetic inheritance, and plant methylation. Plant DNA methylation research reveals methylation patterns and describing variations in plant tissues. Determining the kinetics of DNA methylation in diverse plant tissues is also a new field. However, it is vital to understand regulatory and developmental decisions and use plant model species to develop new commercial crops; that are more resistant to stress and yield more. There are several methods available for assessing DNA methylation data. The performance of several techniques is assessed in A. thaliana, which has a smaller genome than hexaploid bread wheat. Keywords: DNA methylation, plants, process, use and benefits

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Cloning and Characterization of EphA3 (Hek) Gene Promoter: DNA Methylation Regulates Expression in Hematopoietic Tumor Cells

Blood ◽

10.1182/blood.v94.7.2477.419k13_2477_2486 ◽

1999 ◽

Vol 94 (7) ◽

pp. 2477-2486 ◽

Cited By ~ 52

Author(s):

Mirella Dottori ◽

Michelle Down ◽

Andreas Hüttmann ◽

David R. Fitzpatrick ◽

Andrew W. Boyd

Keyword(s):

Dna Methylation ◽

Cell Lines ◽

Tyrosine Kinases ◽

Expression Patterns ◽

Gene Promoter ◽

Restriction Enzymes ◽

Clinical Samples ◽

Cpg Dinucleotides ◽

Normal Tissues

The Eph family of receptor tyrosine kinases (RTK) has restricted temporal and spatial expression patterns during development, and several members are also found to be upregulated in tumors. Very little is known of the promoter elements or regulatory factors required for expression of Eph RTK genes. In this report we describe the identification and characterization of the EphA3 gene promoter region. A region of 86 bp located at −348 bp to −262 bp upstream from the transcription start site was identified as the basal promoter. This region was shown to be active in both EphA3-expressing and -nonexpressing cell lines, contrasting with the widely different levels of EphA3 expression. We noted a region rich in CpG dinucleotides downstream of the basal promoter. Using Southern blot analyses with methylation-sensitive restriction enzymes and bisulfite sequencing of genomic DNA, sites of DNA methylation were identified in hematopoietic cell lines which correlated with their levels of EphA3 gene expression. We showed that EphA3 was not methylated in normal tissues but that a subset of clinical samples from leukemia patients showed extensive methylation, similar to that observed in cell lines. These results suggest that DNA methylation may be an important mechanism regulating EphA3 transcription in hematopoietic tumors.

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