Abstract
Abstract Tissue specific DNA methylation is accountable for regulating gene expression and cellular differentiation during normal human development. Aberrant tissue specific gene regulation may lead to the development of many disease states including cancer. Genome-wide analysis of DNA methylation (methylome) in both healthy and diseased tissue is essential to understand the functional consequence of altered DNA methylation. Acute lymphoblastic leukemia (ALL) is a malignancy associated with precursor B-cells. In order to gain a better understanding of the role of altered DNA methylation in the pathogenesis of ALL, it is essential that we characterize the DNA methylation present in healthy precursor B-cells. Umbilical cord blood is enriched for precursor B-cells and is used as a source of hematopoietic stem cells in the treatment of blood related disorders and malignancies. To study the methylome in healthy precursor B-cells, we have optimized a protocol to isolate 4 subsets of precursor B-cells from umbilical cord blood, and to then generate MBD-seq libraries from the small amount of genomic DNA (< 100ng) isolated from those subsets of precursor B-cells. Subsets of precursor B-cells were isolated from cord blood based on the level of expression of cell surface antigen. Initially mononuclear cells were isolated by Ficoll-plaque followed by magnetic labeling and depletion of non B-cells. B-cells were labeled with antibodies against CD19, CD34 and CD45 surface antigens and sorted by flow cytometry. Based on expression level of surface antigen, four different subsets of precursor B-cells were sorted (CD19+/CD34+; CD19+/CD34-/CD45low; CD19+/CD34-/CD45med; and CD19+/CD34-/CD45high). Immediately after cell sorting, DNA was isolated by using a commercially available kit. The entire amount of DNA from CD19+/CD34+ and CD19+/CD34-/CD45low (<100ng) subsets, and 100ng of DNA from CD19+/CD34-/CD45med and CD19+/CD34-/CD45high subsets were fragmented by sonication using Diagenode Bioruptor. Sequencing libraries were generated using the Illumina Chip-seq protocol with modifications. Enrichment of methylated DNA was validated by PCR amplification of methylated (SLC25A37) and unmethylated (APC) regions. MBD-seq libraries were sequenced using Illumina next-generation sequencing technology and then sequenced data were analyzed by NextGENe® software. We found ∼45 million uniquely matched reads covering ∼700 million bases in the human genome for each precursor B-cell subset. Methylated peaks were identified using the Peak Identification tool in NextGENe. The results demonstrated a differential pattern of DNA methylation in the subsets of healthy precursor B-cells. Citation Format: Md Almamun, Kristen H. Taylor. Genome-wide DNA methylation analysis in subsets of precursor B-cells isolated from umbilical cord blood. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5365. doi:10.1158/1538-7445.AM2013-5365
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