Cooperative Effects of Cytosine Methylation on DNA Structure and Dynamics
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The behavior of the structural parameters of DNA considering different levels of methylation in CpG islands is studied by means of full-atom molecular dynamics simulations and electronic circular dichroism, both in an artificial model system and in a gene promoter sequence. It is demonstrated that methylation although intrinsically brings quite local perturbations may, if its level is high enough, induce cooperative effects that strongly modify the DNA backbone torsional parameters altering the helicity as compared to the nonmethylated case. Because methylation of the CpG island is correlated with the regulation of gene expression, understanding the structural modifications induced in DNA is crucial to characterize all the fine equilibria into play in epigenetics phenomena.Keywords:
CpG site
Cytosine
Epigenomics
Dynamics
The p73 gene is a member of the p53 tumor suppressor gene family. The p73 gene contains a distal P1 promoter and a proximal P2 promoter. The P1 promoter is responsible for expression of the TAp73 protein isoform containing a transactivation domain (TA). The ΔNp73 protein isoform is expressed from the P2 promoter and lacks the N‐terminal TA domain. In this study, we analyzed six CpG islands in these p73 promoter regions. The purpose of this project was to quantitatively assess the degree of methylation at each CpG site in the six CpG islands. Three prostate cancer (PCa) cell lines (DU145, PC‐3, LNCaP) and non‐cancerous primary prostate cells were cultured, and cell pellets were collected. Cellular genomic DNAs were subjected to pyrosequencing analyses of five CpG islands (CpG islands #1–5) located in the P1 promoter region and one CpG island (CpG island #6) in the P2 promoter region. In total, the degree of methylation was determined for 241 individual CpG sites. Pyrosequencing analysis of CpG island #1 indicated 15 of 23 CpG sites were methylated >90% in all four cell types. Seven CpG sites had a methylation frequency range of 71.1% to 96.5%. The remaining CpG site methylation ranged between 60% to 68.8% across the four cell types studied. In CpG island #2, 9 of the 14 CpG sites were methylated >91.9% in all four cell types studied, and the 5 remaining CpG sites ranged from 70.3% to 95.6% methylation. In CpG island #3, the 37 CpG sites were methylated <6.4% in all cell types studied. In CpG island #4, none of the 112 CpG sites were methylated >64.7% in primary cells. Differential methylation was observed in the 112 CpG sites among the three PCa cell lines, ranging from 0.0% up to 91.8%. In CpG island #5, the 45 CpG sites in primary cells were methylated <43.19 %, but PCa cells displayed differential methylation values from 0.0% up to 92.7%. In CpG island #6, the 11 CpG sites were methylated <9.0% in primary cells, whereas, PCa cell lines were differentially methylated from 0.1% up to 98.1%. Of the six CpG islands analyzed, CpG island #3 had the lowest degree of methylation in all samples analyzed. CpG islands #1 and #2 had high degrees of CpG methylation in all four cell types. Differential methylation was observed in CpG island #4, CpG island #5, and CpG island #6. Generally, CpG sites were methylated to higher degrees in PCa cell lines compared to primary cells. Furthermore, the overall trend in high frequency CpG methylation decreased from DU145 to PC‐3 to LNCaP. We are currently performing chromatin immunoprecipitation (ChIP) assays in the differentially methylated regions in order to identify potentially important transcription factors. Support or Funding Information University of Tampa Department of Chemistry, Biochemistry & Physics Summer Research Fellowship Program This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
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Bisulfite sequencing
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Epigenomics-Based DiagnosticsThe term "epigenetics," first coined in 1942, refers to heritable traits of cells (over many rounds of cell division) that do not involve changes to the underlying DNA sequence.The 2 predominant epigenetic mechanisms are DNA methylation and histone modification.
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Cancer Epigenetics
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The regulatory role of cytosine methylation in the genome is incompletely understood despite considerable evidence that it is often associated with the repression of specific gene expression (Doerfler, 1983; Cedar, 1988). The emerging data on distribution of the CpG dinucleotide, which is the major target for cytosine methylation, is thought to reflect a basic type of organization of regulatory sequences in the genome. This dinucleotide is severely underrepresented in bulk vertebrate DNA; but it occurs at the frequency predicted by base composition in CpG islands (Bird, 1986). It is estimated that from 15% to 30% of known genes contain CpG islands. Although they can be found in the 3′ flanking region and in the transcriptional unit of associated genes, a large proportion occur in the 5′ flanking regions which overlap with promoter regions (Gardiner-Garden and Frommer, 1987). Since CpG island sequences are potentially targets for methylation, it is not clear why some of these regions cannot be methylated in the cell (Lock et al., 1986; Dobkin et al., 1989; Szyf et al., 1990) and if, functionally, cytosine methylation of CpG islands in promoter region inactivates transcription.
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DNA methylation is a major epigenetic mark with important roles in genetic regulation. Methylated cytosines are found primarily at CpG dinucleotides, but are also found at non-CpG sites (CpA, CpT, and CpC). The general functions of CpG and non-CpG methylation include gene silencing or activation depending on the methylated regions. CpG and non-CpG methylation are found throughout the whole genome, including repetitive sequences, enhancers, promoters, and gene bodies. Interestingly, however, non-CpG methylation is restricted to specific cell types, such as pluripotent stem cells, oocytes, neurons, and glial cells. Thus, accumulation of methylation at non-CpG sites and CpG sites in neurons seems to be involved in development and disease etiology. Here, we provide an overview of CpG and non-CpG methylation and their roles in neurological diseases.
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RNA-Directed DNA Methylation
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The dynamic changes and structural patterns of DNA methylation of genes without CpG islands are poorly characterized. The relevance of CpG to the non-CpG methylation equilibrium in transcriptional repression is unknown. In this work, we analyzed the DNA methylation pattern of the 5'-flanking of the myogenin gene, a positive regulator of muscle differentiation with no CpG island and low CpG density, in both C2C12 muscle satellite cells and embryonic muscle. Embryonic brain was studied as a non-expressing tissue. High levels of both CpG and non-CpG methylation were observed in non-expressing experimental conditions. Both CpG and non-CpG methylation rapidly dropped during muscle differentiation and myogenin transcriptional activation, with an active demethylation dynamics. Non-CpG demethylation occurred more rapidly than CpG demethylation. Demethylation spread from initially highly methylated short CpC-rich elements to a virtually unmethylated status. These short elements have a high CpC content and density, share some motifs and largely coincide with putative recognition sequences of some differentiation-related transcription factors. Our findings point to a dynamically controlled equilibrium between CpG and non-CpG active demethylation in the transcriptional control of tissue-specific genes. The short CpC-rich elements are new structural features of the methylation machinery, whose functions may include priming the complete demethylation of a transcriptionally crucial DNA region.
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AbstractMethylation of CpG-dinucleotides within proximal promoters is often associated with transcriptional silencing. Methylation-dependent repression is well established for hypermethylated CpG-island promoters that are characterized by a high density of CpG residues. The effect of CpG DNA methylation on CpG-poor promoters is less well characterized, probably due to the lack of convenient assay systems to test promoter activities in vitro. In this report, we describe a novel luciferase reporter vector, pCpGL, which completely lacks CpG dinucleotides and can be used to study the effect of promoter DNA methylation in transfection assays. Whereas a traditional reporter vector that contains a large number of backbone CpG residues significantly represses a CpG-free promoter when methylated, our new reporter vector is only repressed due to the presence of functionally important, methylated CpG residues. The pCpGL vector provides a useful tool to study the effects of CpG-methylation on CpG-rich and CpG-poor promoters.
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Differentially methylated regions
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Methylation of DNA is one of the mechanisms controlling the expression landscape of the genome. Its pattern is altered in cancer and often results in the hypermethylation of the promoter regions and abnormal expression of tumour suppressor genes. Methylation of CpG dinucleotides located in the binding sites of transcription factors may contribute to the development of cancers by preventing their binding or altering their specificity. We studied the effects of CpG methylation on DNA recognition by the tumour suppressor p53, a transcription factor involved in the response to carcinogenic stress. p53 recognises a large number of DNA sequences, many of which contain CpG dinucleotides. We systematically substituted a CpG dinucleotide at each position in the consensus p53 DNA binding sequence and identified substitutions tolerated by p53. We compared the binding affinities of methylated versus non-methylated sequences by fluorescence anisotropy titration. We found that binding of p53 was not affected by cytosine methylation in a majority of cases. However, for a few sequences containing multiple CpG dinucleotides, such as sites in the RB and Met genes, methylation resulted in a four- to sixfold increase in binding of p53. This approach can be used to quantify the effects of CpG methylation on the DNA recognition by other DNA-binding proteins.
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This paper performed the bioinformatic and experimental studies on the methylation and transcription factor(TF) binding sites(TFBS) of the promoter region of ASPP2 gene.The results revealed that there was a 1391-bp CpG island in this region,which containsed 37 TFBSs of 19 TFs.Morover,23 binding sites of 13 TFs contained CpG dinucleotide and 78% of them(18 sites) were predicted methylated.Therefore,ASPP2 was deduced as a typical CpG-island gene regulated by multiple TFs and the abnormal methylation in this CpG island could interfere the binding of TFs and down regulate the expression of ASPP2.The experimental detection of the methylation of a DNA fragment containing 4 E2F-binding sites in this CpG island of human hepatocarcinoma cell HepG2 and human fibroblast cell HFL-1 verified this bioinformatic prediction.This study demonstrate that the bioinformatic prediction of DNA methylation and TFBS in the promoter region of important genes can provide directions for the related wet experimental studies.
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Illumina Methylation Assay
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Methylation of CpG islands spanning promoter regions is associated with control of gene expression. However, it is considered that methylation of exonic CpG islands without promoter is not related to gene expression, because such exonic CpG islands are usually distant from the promoter. Whether methylation of exonic CpG islands near the promoter, as in the case of a CpG-rich intronless gene, causes repression of the promoter remains unknown. To gain insight into this issue, we investigated the distribution and methylation status of CpG dinucleotides in the mouse Tact1/Actl7b gene, which is intronless and expressed exclusively in testicular germ cells. The region upstream to the gene was poor in CpG, with CpG dinucleotides absent from the core promoter. However, a CpG island was found inside the open reading frame (ORF). Analysis of the methylation status of the Tact1/Actl7b gene including the 5'-flanking area demonstrated that all CpG sites were methylated in somatic cells, whereas these sites were unmethylated in the Tact1/Actl7b-positive testis. Trans fection experiments with in vitro-methylated constructs indicated that methylation of the ORF but not 5' upstream repressed Tact1/Actl7b promoter activity in somatic cells. Similar effects of ORF methylation on the promoter activity were observed in testicular germ cells. These are the first results indicating that methylation of the CpG island in the ORF represses its promoter in somatic cells and demethylation is necessary for gene expression in spermatogenic cells.
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Differentially methylated regions
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