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Boyle, Patrick

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Boyle

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Patrick

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Boyle, Patrick

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2012 - 20132

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Author's Publications: search.filters.isAuthorOfPublication.c5141872-46ce-4d52-ba3d-b95d2f6730ce

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    Gel-free multiplexed reduced representation bisulfite sequencing for large-scale DNA methylation profiling
    (BioMed Central, 2012) Boyle, Patrick; Clement, Kendell; Gu, Hongcang; Smith, Zachary; Ziller, Michael; Fostel, Jennifer L; Holmes, Laurie; Meldrim, Jim; Kelley, Fontina; Gnirke, Andreas; Meissner, Alexander
    Sequencing-based approaches have led to new insights about DNA methylation. While many different techniques for genome-scale mapping of DNA methylation have been employed, throughput has been a key limitation for most. To further facilitate the mapping of DNA methylation, we describe a protocol for gel-free multiplexed reduced representation bisulfite sequencing (mRRBS) that reduces the workload dramatically and enables processing of 96 or more samples per week. mRRBS achieves similar CpG coverage to the original RRBS protocol, while the higher throughput and lower cost make it better suited for large-scale DNA methylation mapping studies, including cohorts of cancer samples.
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    Genome-wide Map of Quantified Epigenetic Changes during In vitro Chondrogenic Differentiation of Primary Human Mesenchymal Stem Cells
    (BioMed Central, 2013) Herlofsen, Sarah; Bryne, Jan Christian; Høiby, Torill; Wang, Li; Issner, Robbyn; Zhang, Xiaolan; Coyne, Michael; Boyle, Patrick; Gu, Hongcang; Meza-Zepeda, Leonardo A; Collas, Philippe; Mikkelsen, Tarjei S.; Brinchmann, Jan E
    Background: For safe clinical application of engineered cartilage made from mesenchymal stem cells (MSCs), molecular mechanisms for chondrogenic differentiation must be known in detail. Changes in gene expression and extracellular matrix synthesis have been extensively studied, but the epigenomic modifications underlying these changes have not been described. To this end we performed whole-genome chromatin immunoprecipitation and deep sequencing to quantify six histone modifications, reduced representation bisulphite sequencing to quantify DNA methylation and mRNA microarrays to quantify gene expression before and after 7 days of chondrogenic differentiation of MSCs in an alginate scaffold. To add to the clinical relevance of our observations, the study is based on primary bone marrow-derived MSCs from four donors, allowing us to investigate inter-individual variations. Results: We see two levels of relationship between epigenetic marking and gene expression. First, a large number of genes ontogenetically linked to MSC properties and the musculoskeletal system are epigenetically prepatterned by moderate changes in H3K4me3 and H3K9ac near transcription start sites. Most of these genes remain transcriptionally unaltered. Second, transcriptionally upregulated genes, more closely associated with chondrogenesis, are marked by H3K36me3 in gene bodies, highly increased H3K4me3 and H3K9ac on promoters and 5' end of genes, and increased H3K27ac and H3K4me1 marking in at least one enhancer region per upregulated gene. Within the 7-day time frame, changes in promoter DNA methylation do not correlate significantly with changes in gene expression. Inter-donor variability analysis shows high level of similarity between the donors for this data set. Conclusions: Histone modifications, rather than DNA methylation, provide the primary epigenetic control of early differentiation of MSCs towards the chondrogenic lineage.