In recent years the Illumina HumanMethylation450 (HM450) BeadChip has provided a user-friendly platform to profile DNA methylation in human samples. However, HM450 lacked coverage of distal ...regulatory elements. Illumina have now released the MethylationEPIC (EPIC) BeadChip, with new content specifically designed to target these regions. We have used HM450 and whole-genome bisulphite sequencing (WGBS) to perform a critical evaluation of the new EPIC array platform.
EPIC covers over 850,000 CpG sites, including >90 % of the CpGs from the HM450 and an additional 413,743 CpGs. Even though the additional probes improve the coverage of regulatory elements, including 58 % of FANTOM5 enhancers, only 7 % distal and 27 % proximal ENCODE regulatory elements are represented. Detailed comparisons of regulatory elements from EPIC and WGBS show that a single EPIC probe is not always informative for those distal regulatory elements showing variable methylation across the region. However, overall data from the EPIC array at single loci are highly reproducible across technical and biological replicates and demonstrate high correlation with HM450 and WGBS data. We show that the HM450 and EPIC arrays distinguish differentially methylated probes, but the absolute agreement depends on the threshold set for each platform. Finally, we provide an annotated list of probes whose signal could be affected by cross-hybridisation or underlying genetic variation.
The EPIC array is a significant improvement over the HM450 array, with increased genome coverage of regulatory regions and high reproducibility and reliability, providing a valuable tool for high-throughput human methylome analyses from diverse clinical samples.
Mapping genomic and epigenomic evolution in cancer ecosystems Ushijima, Toshikazu; Clark, Susan J; Tan, Patrick
Science (American Association for the Advancement of Science),
2021-Sep-24, 2021-09-24, 20210924, Letnik:
373, Številka:
6562
Journal Article
Recenzirano
Cancer is a major cause of global mortality underpinned by genomic and epigenomic derangements. Here, we highlight the importance of multimodal data integration in understanding the molecular ...evolution of malignant cell states across the cancer life cycle. The widespread presence of driver mutations and epigenetic alterations in normal-appearing tissues is prompting a reassessment of how cancer initiation is defined. In later-stage cancers, studying the roles of clonal selection, epigenomic adaptation, and persister cells in metastasis and therapy resistance is an emerging field. Finally, the importance of tumor ecosystems in driving cancer development is being unraveled by single-cell and spatial technologies at unprecedented resolution. Improving cancer risk assessment and accelerating therapeutic discovery for patients will require robust, comprehensive, and integrated temporal, spatial, and multilevel tumor atlases across the cancer life cycle.
How DNA methylation is interpreted and influences genome regulation remains largely unknown. Proteins of the methyl-CpG-binding domain (MBD) family are primary candidates for the readout of DNA ...methylation as they recruit chromatin remodelers, histone deacetylases and methylases to methylated DNA associated with gene repression. MBD protein binding requires both functional MBD domains and methyl-CpGs; however, some MBD proteins also bind unmethylated DNA and active regulatory regions via alternative regulatory domains or interaction with the nucleosome remodeling deacetylase (NuRD/Mi-2) complex members. Mutations within MBD domains occur in many diseases, including neurological disorders and cancers, leading to loss of MBD binding specificity to methylated sites and gene deregulation. Here, we summarize the current state of knowledge about MBD proteins and their role as readers of the epigenome.
The identification and characterisation of differentially methylated regions (DMRs) between phenotypes in the human genome is of prime interest in epigenetics. We present a novel method, DMRcate, ...that fits replicated methylation measurements from the Illumina HM450K BeadChip (or 450K array) spatially across the genome using a Gaussian kernel. DMRcate identifies and ranks the most differentially methylated regions across the genome based on tunable kernel smoothing of the differential methylation (DM) signal. The method is agnostic to both genomic annotation and local change in the direction of the DM signal, removes the bias incurred from irregularly spaced methylation sites, and assigns significance to each DMR called via comparison to a null model.
We show that, for both simulated and real data, the predictive performance of DMRcate is superior to those of Bumphunter and Probe Lasso, and commensurate with that of comb-p. For the real data, we validate all array-derived DMRs from the candidate methods on a suite of DMRs derived from whole-genome bisulfite sequencing called from the same DNA samples, using two separate phenotype comparisons.
The agglomeration of genomically localised individual methylation sites into discrete DMRs is currently best served by a combination of DM-signal smoothing and subsequent threshold specification. The findings also suggest the design of the 450K array shows preference for CpG sites that are more likely to be differentially methylated, but its overall coverage does not adequately reflect the depth and complexity of methylation signatures afforded by sequencing. For the convenience of the research community we have created a user-friendly R software package called DMRcate, downloadable from Bioconductor and compatible with existing preprocessing packages, which allows others to apply the same DMR-finding method on 450K array data.
The Sociocultural Attitudes Towards Appearance Questionnaire-3 (SATAQ-3) and its earlier versions are measures designed to assess societal and interpersonal aspects of appearance ideals. ...Correlational, structural equation modeling, and prospective studies of the SATAQ-3 have shown consistent and significant associations with measures of body image disturbance and eating pathology. In the current investigation, the SATAQ-3 was revised to improve upon some conceptual limitations and was evaluated in 4 U.S. and 3 international female samples, as well as a U.S. male sample. In Study 1, exploratory and confirmatory factor analyses for a sample of women from the Southeastern United States (N = 859) indicated a 22-item scale with 5 factors: Internalization: Thin/Low Body Fat, Internalization: Muscular/Athletic, Pressures: Family, Pressures: Media, Pressures: Peers. This scale structure was confirmed in 3 independent and geographically diverse samples of women from the United States (East Coast N = 440, West Coast N = 304, and North/Midwest N = 349). SATAQ-4 scale scores demonstrated excellent reliability and good convergent validity with measures of body image, eating disturbance, and self-esteem. Study 2 replicated the factorial validity, reliability, and convergent validity of the SATAQ-4 in an international sample of women drawn from Italy, England, and Australia (N = 362). Study 3 examined a sample of college males from the United States (N = 271); the 5-factor solution was largely replicated, yet there was some evidence of an underlying structure unique to men. Future research avenues include additional item testing and modification of the scale for men, as well as adaptation of the measure for children and adolescents.
H2A.Z is a histone variant that provides specific structural and docking-side properties to the nucleosome, resulting in diverse and specialised molecular and cellular functions. In this review, we ...discuss the latest studies uncovering new functional aspects of mammalian H2A.Z in gene transcription, including pausing and elongation of RNA polymerase II (RNAPII) and enhancer activity; DNA repair; DNA replication; and 3D chromatin structure. We also review the recently described role of H2A.Z in embryonic development, cell differentiation, neurodevelopment, and brain function. In conclusion, our cumulative knowledge of H2A.Z over the past 40 years, in combination with the implementation of novel molecular technologies, is unravelling an unexpected and complex role of histone variants in gene regulation and disease.
DNA methylation patterns are altered in numerous diseases and often correlate with clinically relevant information such as disease subtypes, prognosis and drug response. With suitable assays and ...after validation in large cohorts, such associations can be exploited for clinical diagnostics and personalized treatment decisions. Here we describe the results of a community-wide benchmarking study comparing the performance of all widely used methods for DNA methylation analysis that are compatible with routine clinical use. We shipped 32 reference samples to 18 laboratories in seven different countries. Researchers in those laboratories collectively contributed 21 locus-specific assays for an average of 27 predefined genomic regions, as well as six global assays. We evaluated assay sensitivity on low-input samples and assessed the assays' ability to discriminate between cell types. Good agreement was observed across all tested methods, with amplicon bisulfite sequencing and bisulfite pyrosequencing showing the best all-round performance. Our technology comparison can inform the selection, optimization and use of DNA methylation assays in large-scale validation studies, biomarker development and clinical diagnostics.
Epigenetic gene deregulation in cancer commonly occurs through chromatin repression and promoter hypermethylation of tumor-associated genes. However, the mechanism underpinning epigenetic-based gene ...activation in carcinogenesis is still poorly understood. Here, we identify a mechanism of domain gene deregulation through coordinated long-range epigenetic activation (LREA) of regions that typically span 1 Mb and harbor key oncogenes, microRNAs, and cancer biomarker genes. Gene promoters within LREA domains are characterized by a gain of active chromatin marks and a loss of repressive marks. Notably, although promoter hypomethylation is uncommon, we show that extensive DNA hypermethylation of CpG islands or “CpG-island borders” is strongly related to cancer-specific gene activation or differential promoter usage. These findings have wide ramifications for cancer diagnosis, progression, and epigenetic-based gene therapies.
► We identify an epigenetic-based mechanism of regional cancer gene activation ► Long-range epigenetic activation affects multiple adjacent tumor-associated genes ► Regions are characterized by gain of active chromatin marks and loss of repressive marks ► Promoter hypermethylation relates to cancer gene activation and changes in promoter usage
The architectural protein CTCF is a mediator of chromatin conformation, but how CTCF binding to DNA is orchestrated to maintain long-range gene expression is poorly understood. Here we perform RNAi ...knockdown to reduce CTCF levels and reveal a shared subset of CTCF-bound sites are robustly resistant to protein depletion. The 'persistent' CTCF sites are enriched at domain boundaries and chromatin loops constitutive to all cell types. CRISPR-Cas9 deletion of 2 persistent CTCF sites at the boundary between a long-range epigenetically active (LREA) and silenced (LRES) region, within the Kallikrein (KLK) locus, results in concordant activation of all 8 KLK genes within the LRES region. CTCF genome-wide depletion results in alteration in Topologically Associating Domain (TAD) structure, including the merging of TADs, whereas TAD boundaries are not altered where persistent sites are maintained. We propose that the subset of essential CTCF sites are involved in cell-type constitutive, higher order chromatin architecture.
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