Comprehensive profiling of zebrafish hepatic proximal promoter CpG island methylation and its modification during chemical carcinogenesis

Background\ud DNA methylation is an epigenetic mechanism associated with regulation of gene expression and it is modulated during chemical carcinogenesis. The zebrafish is increasingly employed as a human disease model; however there is a lack of information on DNA methylation in zebrafish and during fish tumorigenesis. \ud \ud Results\ud A novel CpG island tiling array containing 44,000 probes, in combination with immunoprecipitation of methylated DNA, was used to achieve the first comprehensive methylation profiling of normal adult zebrafish liver. DNA methylation alterations were detected in zebrafish liver tumors induced by the environmental carcinogen 7, 12-dimethylbenz(a)anthracene. Genes significantly hypomethylated in tumors were associated particularly with proliferation, glycolysis, transcription, cell cycle, apoptosis, growth and metastasis. Hypermethylated genes included those associated with anti-angiogenesis and cellular adhesion. Of 49 genes that were altered in expression within tumors, and which also had appropriate CpG islands and were co-represented on the tiling array, approximately 45% showed significant changes in both gene expression and methylation. \ud \ud Conclusion\ud The functional pathways containing differentially methylated genes in zebrafish hepatocellular carcinoma have also been reported to be aberrantly methylated during tumorigenesis in humans. These findings increase the confidence in the use of zebrafish as a model for human cancer in addition to providing the first comprehensive mapping of DNA methylation in the normal adult zebrafish liver. \ud \u

Artificial escape from XCI by DNA methylation editing of the CDKL5 gene.

A significant number of X-linked genes escape from X chromosome inactivation and are associated with a distinct epigenetic signature. One epigenetic modification that strongly correlates with X-escape is reduced DNA methylation in promoter regions. Here, we created an artificial escape by editing DNA methylation on the promoter of CDKL5, a gene causative for an infantile epilepsy, from the silenced X-chromosomal allele in human neuronal-like cells. We identify that a fusion of the catalytic domain of TET1 to dCas9 targeted to the CDKL5 promoter using three guide RNAs causes significant reactivation of the inactive allele in combination with removal of methyl groups from CpG dinucleotides. Strikingly, we demonstrate that co-expression of TET1 and a VP64 transactivator have a synergistic effect on the reactivation of the inactive allele to levels >60% of the active allele. We further used a multi-omics assessment to determine potential off-targets on the transcriptome and methylome. We find that synergistic delivery of dCas9 effectors is highly selective for the target site. Our findings further elucidate a causal role for reduced DNA methylation associated with escape from X chromosome inactivation. Understanding the epigenetics associated with escape from X chromosome inactivation has potential for those suffering from X-linked disorders

Genome-wide DNA methylation detection by MethylCap-seq and Infinium HumanMethylation450 BeadChips: an independent large-scale comparison.

Two cost-efficient genome-scale methodologies to assess DNA-methylation are MethylCap-seq and Illumina's Infinium HumanMethylation450 BeadChips (HM450). Objective information regarding the best-suited methodology for a specific research question is scant. Therefore, we performed a large-scale evaluation on a set of 70 brain tissue samples, i.e. 65 glioblastoma and 5 non-tumoral tissues. As MethylCap-seq coverages were limited, we focused on the inherent capacity of the methodology to detect methylated loci rather than a quantitative analysis. MethylCap-seq and HM450 data were dichotomized and performances were compared using a gold standard free Bayesian modelling procedure. While conditional specificity was adequate for both approaches, conditional sensitivity was systematically higher for HM450. In addition, genome-wide characteristics were compared, revealing that HM450 probes identified substantially fewer regions compared to MethylCap-seq. Although results indicated that the latter method can detect more potentially relevant DNA-methylation, this did not translate into the discovery of more differentially methylated loci between tumours and controls compared to HM450. Our results therefore indicate that both methodologies are complementary, with a higher sensitivity for HM450 and a far larger genome-wide coverage for MethylCap-seq, but also that a more comprehensive character does not automatically imply more significant results in biomarker studies

A functional data analytic approach for region level differential DNA methylation detection

DNA methylation is an epigenetic modification that can alter gene expression without a DNA sequence change. The role of DNA methylation in biological processes and human health is important to understand, with many studies identifying associations between specific methylation patterns and diseases such as cancer. In mammals, DNA methylation almost always occurs when a methyl group attaches to a cytosine followed by a guanine (i.e. CpG dinucleotides) on the DNA sequence. Many statistical methods have been developed to test for a difference in DNA methylation levels between groups (e.g. healthy vs disease) at individual cytosines. Site level testing is often followed by a post hoc aggregation procedure that explores regional differences. Although analyzing CpGs individually provides useful information, there are both biological and statistical reasons to test entire genomic regions for differential methylation. The individual loci may be noisy but the overall regions tend to be informative. Also, the biological function of regions is better studied and are more correlated to gene expression, so the interpretation of results will be more meaningful for region-level tests. This study focuses on developing two techniques, functional principal component analysis (FPCA) and smoothed functional principal component analysis (SFPCA), to identify differentially methylated regions (DMRs) that will enable discovery of epigenomic structural variations in NGS data. Using real and simulated data, the performance of these novel approaches are compared with an alternative method (M3D) for region level testing --Abstract, page iv

Strategies for analyzing bisulfite sequencing data

DNA methylation is one of the main epigenetic modifications in the eukaryotic genome and has been shown to play a role in cell-type specific regulation of gene expression, and therefore cell-type identity. Bisulfite sequencing is the gold-standard for measuring methylation over the genomes of interest. Here, we review several techniques used for the analysis of high-throughput bisulfite sequencing. We introduce specialized short-read alignment techniques as well as pre/post-alignment quality check methods to ensure data quality. Furthermore, we discuss subsequent analysis steps after alignment. We introduce various differential methylation methods and compare their performance using simulated and real bisulfite-sequencing datasets. We also discuss the methods used to segment methylomes in order to pinpoint regulatory regions. We introduce annotation methods that can be used further classification of regions returned by segmentation or differential methylation methods. Lastly, we review software packages that implement strategies to efficiently deal with large bisulfite sequencing datasets locally and also discuss online analysis workflows that do not require any prior programming skills. The analysis strategies described in this review will guide researchers at any level to the best practices of bisulfite sequencing analysis

개의 유선암과 인간의 유방암 모두에서의 ANK2의 과 메틸화

학위논문 (석사) -- 서울대학교 대학원 : 수의과대학 수의학과, 2021. 2. 조제열.개의 유선암 (canine mammary gland tumor)은 사람 여성의 유방암과 같이 암컷 개에서 가장 흔히 발견되는 암이다. 개의 유선암과 사람의 유방암에 존재하는 여러 유사성 때문에 개의 유선암을 연구하는 것은 수의학에 국한된 것이 아니라 사람의 유방암을 이해하기 위한 비교의학적 측면에서 매우 중요하다. DNA 메틸화를 조직 및 액체생검에서 생체 표지자로 사용하는 시도는 많이 진행되고 있지만, 개의 유선암에 대한 연구는 매우 제한적으로 이루어지고 있다. 이 연구에서, 우리는 개의 유선암 조직의 메틸롬(Methylome) 분석을 통해 ANK2 및 EPAS 유전자의 인트론 영역에서 정상 조직과 다른 차등 메틸화 영역(Differentially methylated regions, DMGs)을 발견하였다. 또한, 이 두 지역의 차등 메틸화를 조직뿐만 아니라 환자유래 혈장에 존재하는 순환 유리 DNA (Cell free DNA, cfDNA)로부터 정량 할 수 있는 정량적 메틸화 특이적 PCR(quantitative methylation specific PCR, qMSP) 방법을 확립하였다. 이 방법을 통해 우리는 두 영역의 유선암 특이 과 메틸화를 추가된 조직 생검 시료를 통해 확인하였다. 나아가, ANK2인트론 영역은 조직 생검 시료뿐만 아니라 유선암 환자 유래 혈장에 존재하는 cfDNA에서도 유의한 메틸화를 보였다. 이 결과는 ANK2 및 EPAS의 인트론 영역의 메틸화가 유선암의 조직은 물론 액체생검을 위한 생체표지자로 사용될 수 있음을 의미한다. 흥미롭게도, 우리는 이러한 ANK2의 개유선암 특이 과메틸화는 비교 의학적인 측면에서 사람의 유방암에서도 과메틸화 되어있는 경향을 확인할 수 있었다. 이 연구 결과는 비교의학적 접근이 가지는 장점을 잘 보여주며, 이를 이용한 실제 임상적용을 위한 활용가능성을 높여준다.Canine Mammary Tumors (CMT) constitute the most common tumor types found in female dogs. Understanding this cancer through extensive research is important not only for clinical veterinary applications, but also in the scope of comparative oncology. The use of DNA methylation as a biomarker has been noted for numerous cancers in the form of both tissue and liquid biopsies, yet the study of methylation in CMT has been limited. By analyzing our canine Methyl-binding domain sequencing (MBD-seq) data, we identified intron regions of canine ANK2 and EPAS1 as differentially methylated regions (DMGs) in CMT. Subsequently, we established quantitative Methylation Specific PCR (qMSP) of ANK2 and EPAS1 to validate the target hypermethylation in CMT tissue, as well as cell free DNA (cfDNA) from CMT plasma. Both ANK2 and EPAS1 were hypermethylated in CMT and highlighted as potential tissue biomarkers in CMT. ANK2 additionally showed significant hypermethylation in the plasma cfDNA of CMT, indicating that it could be a potential liquid biopsy biomarker as well. A similar trend towards hypermethylation was indicated in HBC at a specific CpG of the ANK2 target on the orthologous human region, which validates the comparative approach using aberrant methylation in CMT.1. Introduction 9 2. Results 12 2.1 Identification of differentially methylated region 2.2 Evaluation of differentially methylated regions in CMT and adjacent normal tissue 2.3 Detection of differential methylation in canine plasma cfDNA 2.4 Orthologous human regions analyzed from TCGA data 3. Discussion 33 4. Materials and Methods 41 4.1 Ethics 4.2 Tissue and plasma samples 4.3 Correlation analysis between methylation and gene expression 4.4 DNA isolation 4.5 Bisulfite Sequencing 4.6 Quantitative Methylation-Specific PCR (qMSP) 4.7 Human TCGA Data Bibliography 53Maste

Strategies for analyzing bisulfite sequencing data

DNA methylation is one of the main epigenetic modifications in the eukaryotic genome; it has been shown to play a role in cell-type specific regulation of gene expression, and therefore cell-type identity. Bisulfite sequencing is the gold-standard for measuring methylation over the genomes of interest. Here, we review several techniques used for the analysis of high-throughput bisulfite sequencing. We introduce specialized short-read alignment techniques as well as pre/post-alignment quality check methods to ensure data quality. Furthermore, we discuss subsequent analysis steps after alignment. We introduce various differential methylation methods and compare their performance using simulated and real bisulfite sequencing datasets. We also discuss the methods used to segment methylomes in order to pinpoint regulatory regions. We introduce annotation methods that can be used for further classification of regions returned by segmentation and differential methylation methods. Finally, we review software packages that implement strategies to efficiently deal with large bisulfite sequencing datasets locally and we discuss online analysis workflows that do not require any prior programming skills. The analysis strategies described in this review will guide researchers at any level to the best practices of bisulfite sequencing analysis

Subtype-specific CpG island shore methylation and mutation patterns in 30 breast cancer cell lines

BACKGROUND: Aberrant epigenetic modifications, including DNA methylation, are key regulators of gene activity in tumorigenesis. Breast cancer is a heterogeneous disease, and large-scale analyses indicate that tumor from normal and benign tissues, as well as molecular subtypes of breast cancer, can be distinguished based on their distinct genomic, transcriptomic, and epigenomic profiles. In this study, we used affinity-based methylation sequencing data in 30 breast cancer cell lines representing functionally distinct cancer subtypes to investigate methylation and mutation patterns at the whole genome level. RESULTS: Our analysis revealed significant differences in CpG island (CpGI) shore methylation and mutation patterns among breast cancer subtypes. In particular, the basal-like B type, a highly aggressive form of the disease, displayed distinct CpGI shore hypomethylation patterns that were significantly associated with downstream gene regulation. We determined that mutation rates at CpG sites were highly correlated with DNA methylation status and observed distinct mutation rates among the breast cancer subtypes. These findings were validated by using targeted bisulfite sequencing of differentially expressed genes (n=85) among the cell lines. CONCLUSIONS: Our results suggest that alterations in DNA methylation play critical roles in gene regulatory process as well as cytosine substitution rates at CpG sites in molecular subtypes of breast cancer. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12918-016-0356-2) contains supplementary material, which is available to authorized users