DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs

Headloop suppression PCR and its application to selective amplification of methylated DNA sequences

Selective amplification in PCR is principally determined by the sequence of the primers and the temperature of the annealing step. We have developed a new PCR technique for distinguishing related sequences in which additional selectivity is dependent on sequences within the amplicon. A 5′ extension is included in one (or both) primer(s) that corresponds to sequences within one of the related amplicons. After copying and incorporation into the PCR product this sequence is then able to loop back, anneal to the internal sequences and prime to form a hairpin structure—this structure is then refractory to further amplification. Thus, amplification of sequences containing a perfect match to the 5′ extension is suppressed while amplification of sequences containing mismatches or lacking the sequence is unaffected. We have applied Headloop PCR to DNA that had been bisulphite-treated for the selective amplification of methylated sequences of the human GSTP1 gene in the presence of up to a 10(5)-fold excess of unmethylated sequences. Headloop PCR has a potential for clinical application in the detection of differently methylated DNAs following bisulphite treatment as well as for selective amplification of sequence variants or mutants in the presence of an excess of closely related DNA sequences

Rap2B promotes proliferation, migration and invasion of human breast cancer through calcium-related ERK1/2 signaling pathway

Rap2B, a member of GTP-binding proteins, is widely upregulated in many types of tumors and promotes migration and invasion of human suprarenal epithelioma. However, the function of Rap2B in breast cancer is unknown. Expression of Rap2B was examined in breast cancer cell lines and human normal breast cell line using Western blot analysis. Using the CCK-8 cell proliferation assay, cell cycle analysis, and transwell migration assay, we also elucidated the role of Rap2B in breast cancer cell proliferation, migration, and invasion. Results showed that the expression of Rap2B is higher in tumor cells than in normal cells. Flow cytometry and Western blot analysis revealed that Rap2B elevates the intracellular calcium level and further promotes extracellular signal-related kinase (ERK) 1/2 phosphorylation. By contrast, calcium chelator BAPTM/AM and MEK inhibitor (U0126) can reverse Rap2B-induced ERK1/2 phosphorylation. Furthermore, Rap2B knockdown inhibits cell proliferation, migration, and invasion abilities via calcium related-ERK1/2 signaling. In addition, overexpression of Rap2B promotes cell proliferation, migration and invasion abilities, which could be neutralized by BAPTM/AM and U0126. Taken together, these findings shed light on Rap2B as a therapeutic target for breast cancer

CMRxRecon: An open cardiac MRI dataset for the competition of accelerated image reconstruction

Cardiac magnetic resonance imaging (CMR) has emerged as a valuable diagnostic tool for cardiac diseases. However, a limitation of CMR is its slow imaging speed, which causes patient discomfort and introduces artifacts in the images. There has been growing interest in deep learning-based CMR imaging algorithms that can reconstruct high-quality images from highly under-sampled k-space data. However, the development of deep learning methods requires large training datasets, which have not been publicly available for CMR. To address this gap, we released a dataset that includes multi-contrast, multi-view, multi-slice and multi-coil CMR imaging data from 300 subjects. Imaging studies include cardiac cine and mapping sequences. Manual segmentations of the myocardium and chambers of all the subjects are also provided within the dataset. Scripts of state-of-the-art reconstruction algorithms were also provided as a point of reference. Our aim is to facilitate the advancement of state-of-the-art CMR image reconstruction by introducing standardized evaluation criteria and making the dataset freely accessible to the research community. Researchers can access the dataset at https://www.synapse.org/#!Synapse:syn51471091/wiki/.Comment: 14 pages, 8 figure

Impact of the Genome on the Epigenome Is Manifested in DNA Methylation Patterns of Imprinted Regions in Monozygotic and Dizygotic Twins

One of the best studied read-outs of epigenetic change is the differential expression of imprinted genes, controlled by differential methylation of imprinted control regions (ICRs). To address the impact of genotype on the epigenome, we performed a detailed study in 128 pairs of monozygotic (MZ) and 128 pairs of dizygotic (DZ) twins, interrogating the DNA methylation status of the ICRs of IGF2, H19, KCNQ1, GNAS and the non-imprinted gene RUNX1. While we found a similar overall pattern of methylation between MZ and DZ twins, we also observed a high degree of variability in individual CpG methylation levels, notably at the H19/IGF2 loci. A degree of methylation plasticity independent of the genome sequence was observed, with both local and regional CpG methylation changes, discordant between MZ and DZ individual pairs. However, concordant gains or losses of methylation, within individual twin pairs were more common in MZ than DZ twin pairs, indicating that de novo and/or maintenance methylation is influenced by the underlying DNA sequence. Specifically, for the first time we showed that the rs10732516 [A] polymorphism, located in a critical CTCF binding site in the H19 ICR locus, is strongly associated with increased hypermethylation of specific CpG sites in the maternal H19 allele. Together, our results highlight the impact of the genome on the epigenome and demonstrate that while DNA methylation states are tightly maintained between genetically identical and related individuals, there remains considerable epigenetic variation that may contribute to disease susceptibility

The potential of epigenetic therapy to target the 3D epigenome in endocrine-resistant breast cancer

Three-dimensional (3D) epigenome remodeling is an important mechanism of gene deregulation in cancer. However, its potential as a target to counteract therapy resistance remains largely unaddressed. Here, we show that epigenetic therapy with decitabine (5-Aza-mC) suppresses tumor growth in xenograft models of pre-clinical metastatic estrogen receptor positive (ER+) breast tumor. Decitabine-induced genome-wide DNA hypomethylation results in large-scale 3D epigenome deregulation, including de-compaction of higher-order chromatin structure and loss of boundary insulation of topologically associated domains. Significant DNA hypomethylation associates with ectopic activation of ER-enhancers, gain in ER binding, creation of new 3D enhancer–promoter interactions and concordant up-regulation of ER-mediated transcription pathways. Importantly, long-term withdrawal of epigenetic therapy partially restores methylation at ER-enhancer elements, resulting in a loss of ectopic 3D enhancer–promoter interactions and associated gene repression. Our study illustrates the potential of epigenetic therapy to target ER+ endocrine-resistant breast cancer by DNA methylation-dependent rewiring of 3D chromatin interactions, which are associated with the suppression of tumor growth