Profiling the genome-wide DNA methylation pattern of porcine ovaries using reduced representation bisulfite sequencing

Both 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are important epigenetic modifications in neurodevelopment. However, there is little research examining the genome-wide patterns of 5mC and 5hmC in brain regions of animals under natural high-altitude conditions. We used oxidative reduced representation bisulfite sequencing (oxRRBS) to determine the 5mC and 5hmC sites in the brain, brainstem, cerebellum, and hypothalamus of yak and cattle. We reported the first map of genome-wide DNA methylation and hydroxymethylation in the brain, brainstem, cerebellum, and hypothalamus of yak (living at high altitudes) and cattle. Overall, we found striking differences in 5mC and 5hmC between the hypothalamus and other brain regions in both yak and cattle. Genome-wide profiling revealed that 5mC level decreased and 5hmC level increased in the hypothalamus than in other regions. Furthermore, we identified differentially methylated regions (DMRs) and differentially hydroxymethylated regions (DhMRs), most of which overlapped with each other. Interestingly, transcriptome results for these brain regions also showed distinctive gene levels in the hypothalamus. Finally, differentially expressed genes (DEGs) regulated by DMRs and DhMRs may play important roles in neuromodulation and myelination. Overall, our results suggested that mediation of 5mC and 5hmC on epigenetic regulation may broadly impact the development of hypothalamus and its biological functions.

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Identification of differential genomic DNA Methylation in the hypothalamus of pubertal rat using reduced representation Bisulfite sequencing

Reproductive Biology and Endocrinology ◽

10.1186/s12958-017-0301-2 ◽

2017 ◽

Vol 15 (1) ◽

Cited By ~ 4

Author(s):

Lei Luo ◽

Zhiqiu Yao ◽

Jing Ye ◽

Yuan Tian ◽

Chen Yang ◽

...

Keyword(s):

Dna Methylation ◽

Genomic Dna ◽

Bisulfite Sequencing ◽

Reduced Representation ◽

Reduced Representation Bisulfite Sequencing

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Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling

Nature Protocols ◽

10.1038/nprot.2010.190 ◽

2011 ◽

Vol 6 (4) ◽

pp. 468-481 ◽

Cited By ~ 424

Author(s):

Hongcang Gu ◽

Zachary D Smith ◽

Christoph Bock ◽

Patrick Boyle ◽

Andreas Gnirke ◽

...

Keyword(s):

Dna Methylation ◽

Bisulfite Sequencing ◽

Reduced Representation ◽

Reduced Representation Bisulfite Sequencing ◽

Dna Methylation Profiling ◽

Genome Scale ◽

Methylation Profiling

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High Resolution Genome Wide DNA Methylation Analysis in a Large Trial Group Reveals a Novel Epigenetically Defined Subgroup of Myeloma Patients Characterized By Developmental Gene Hypermethylation

Blood ◽

10.1182/blood.v124.21.2189.2189 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2189-2189

Author(s):

Martin F Kaiser ◽

Alexander Murison ◽

Charlotte Pawlyn ◽

Eileen M Boyle ◽

David C Johnson ◽

...

Keyword(s):

Dna Methylation ◽

High Resolution ◽

Methylation Pattern ◽

Gene Set Enrichment Analysis ◽

Epigenetic Changes ◽

Dna Hypomethylation ◽

Dna Hypermethylation ◽

Epigenetic Programming ◽

Genome Wide ◽

Dna Methylation Pattern

Abstract Introduction Multiple myeloma is a clinically highly heterogeneous disease, which is reflected by both a complex genome and epigenome. Dynamic epigenetic changes are involved at several stages of myeloma biology, such as transformation and disease progression. Our previous genome wide epigenetic analyses identified prognostically relevant DNA hypermethylation at specific tumor suppressor genes (Kaiser MF et al., Blood 2013), indicating that specific epigenetic programming influences clinical behavior. This clinically relevant finding prompted further investigation of the epigenomic structure of myeloma and its interaction with genetic aberrations. Material and Methods Genome wide DNA methylation of CD138-purified myeloma cells from 464 patients enrolled in the NCRI Myeloma XI trial at presentation were analyzed using the high resolution 450k DNA methylation array platform (Illumina). In addition, 4 plasma cell leukemia (PCL) cases (two t(11;14) and two (4;14)) and 7 myeloma cell lines (HMCL) carrying different translocations were analysed. Analyses were performed in R Bioconductor packages after filtering and removal of low quality and non-uniquely mapping probes. Results Variation in genome wide DNA methylation was analyzed using unsupervised hierarchical clustering of the 10,000 most variable probes, which revealed epigenetically defined subgroups of disease. Presence of recurrent IGH translocations was strongly associated with specific epigenetic profiles. All 60 cases with t(4;14) clustered into two highly similar sub-clusters, confirming that overexpression of the H3K36 methyltransferase MMSET in t(4;14) has a defined and specific effect on the myeloma epigenome. Interestingly, HMCLs KMS-11 and LP-1, which carry t(4;14), MM1.S, a t(14;16) cell line with an E1099K MMSET activating mutation as well as two PCLs with t(4;14) all clustered in one sub-clade. The majority (59/85) of t(11;14) cases showed global DNA hypomethylation compared to t(4;14) cases and clustered in one subclade, indicating a epigenetic programming effect associated with CCND1, with a subgroup of t(11;14) cases showing a variable DNA methylation pattern. In addition to translocation-defined subgroups, a small cluster of samples with a distinct epigenetic profile was identified. In total 7 cases with a shared specific DNA methylation pattern (median inter-sample correlation 0.4) were identified. The group was characterized by DNA hypermethylation (4,341 hypermethylated regions vs. 750 hypomethylated regions) in comparison to all other cases. Intersection of regions hypermethylated in this subgroups with ENCODE datasets revealed mapping to poised enhancers and promoters in H1-hESC, indicating functionally relevant epigenetic changes. Gene set enrichment analysis (KEGG) demonstrated enrichment of developmental pathway genes, e.g. Hedgehog signaling (adj p=5x10exp-13), amongst others and all four HOX clusters were differentially methylated in this group. Of note, three of seven cases in this subgroup carried a t(11;14) and all t(11;14) or t(11;14)-like HMCLs clustered closely together with these patient cases, but not with the cluster carrying the majority of t(11;14) myeloma or t(11;14) PCLs. This potentially indicates that t(11;14) HMCL could be derived from a subgroup of patients with specific epigenetic characteristics. Conclusion Our results indicate that the recurrent IGH translocations are fundamentally involved in shaping the myeloma epigenome through either direct upregulation of epigenetic modifiers (e.g. MMSET) or through insufficiently understood mechanisms. However, developmental epigenetic processes seem to independently contribute to the complexity of the epigenome in some cases. This work provides important insights into the spectrum of epigenetic subgroups of myeloma and helps identify subgroups of disease that may benefit from specific epigenetic therapies currently being developed. Disclosures Walker: Onyx Pharmaceuticals: Consultancy, Honoraria.

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Minimally invasive classification of pediatric solid tumors using reduced representation bisulfite sequencing of cell-free DNA: a proof-of-principle study

10.1101/795047 ◽

2019 ◽

Cited By ~ 1

Author(s):

Ruben Van Paemel ◽

Andries De Koker ◽

Charlotte Vandeputte ◽

Lieke van Zogchel ◽

Tim Lammens ◽

...

Keyword(s):

Minimally Invasive ◽

Bisulfite Sequencing ◽

Methylation Pattern ◽

Histopathological Diagnosis ◽

Cell Free Dna ◽

Reduced Representation ◽

Pediatric Solid Tumors ◽

Free Dna ◽

Reduced Representation Bisulfite Sequencing

AbstractIn the clinical management of pediatric solid tumors, histological examination of tumor tissue obtained by a biopsy remains the gold standard to establish a conclusive pathological diagnosis. The DNA methylation pattern of a tumor is known to correlate with the histopathological diagnosis across cancer types and is showing promise in the diagnostic workup of tumor samples. This methylation pattern can be detected in the cell-free DNA. Here, we provide proof-of-concept of histopathologic classification of pediatric tumors using cell-free reduced representation bisulfite sequencing (cf-RRBS) from retrospectively collected plasma and cerebrospinal fluid samples. We determined the correct tumor type in 49 out of 60 (81.6%) samples starting from minute amounts (less than 10 ng) of cell-free DNA. We demonstrate that the majority of misclassifications were associated with sample quality and not with the extent of disease. Our approach has the potential to help tackle some of the remaining diagnostic challenges in pediatric oncology in a cost-effective and minimally invasive manner.Translational relevanceObtaining a correct diagnosis in pediatric oncology can be challenging in some tumor types, especially in renal tumors or central nervous system tumors. Furthermore, the diagnostic odyssey can result in anxiety and discomfort for these children. By applying a novel technique, reduced representation bisulfite sequencing on cell-free DNA (cf-RRBS), we show the feasibility of obtaining the histopathological diagnosis with a minimally invasive test on either plasma or cerebrospinal fluid. Furthermore, we were able to derive the copy number profile or tumor subtype from the same assay. Given that primary tumor material might be difficult to obtain, in particular in critically ill children or depending on the tumor location, and might be limited in terms of quantity or quality, our assay could become complementary to the classical tissue biopsy in difficult cases.

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