Chromatin Profiles of Human Cells in Health and Disease Using FAIRE Public Deposited

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Last Modified
  • March 20, 2019
Creator
  • Giresi, Paul
    • Affiliation: College of Arts and Sciences, Department of Biology
Abstract
  • Breast cancer is a heterogenous disease comprised of molecularly distinct subtypes with diverse clinical outcomes. Understanding the molecular composition of each subtype will aid in the effective diagnosis and treatment of breast cancer. The composition and activity of subtype-selective regulatory pathways operate, in part, through binding of proteins at distinct sites throughout the genome, often referred to as regulatory elements, to govern levels of gene expression. One of the characteristics of these binding events is the displacement of nucleosomes. Here we have developed a technique called FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements), which is capable of the genome-wide identification of active regulatory elements in human cells based on the nucleosome-depleted nature of these sites. Using FAIRE we have identified the genome-wide set of active regulatory elements in the luminal and basal-like tumor subtypes. Here most of the active regulatory elements were distinct to each subtype and tended to occur not at transcriptional start sites, but at distal regulatory elements. Many of these unique sites also reflected the activity of the regulatory mechanisms present in a given subtype. For example, in the hormone-responsive luminal cells we detected strong FAIRE signals at estrogen-receptor alpha binding sites, whereas the signals are diminished or absent in the hormone nonresponsive basal-like cells. These distal regulatory elements tended to be clustered to form distinct genomic domains containing the set of all expressed genes in the respective subtype, regardless of whether the gene was differentially expressed between the subtypes. This suggests that the combination of gene expression and the subtype-selective active regulatory elements provides an expanded understanding of the molecular complexity between subtypes. The subtype-selective regulatory elements were also enriched with sequence motifs for DNA-binding proteins, which included factors known to be active in the respective subtypes. The remaining sequence motifs should serve as a useful starting point for the identification of additional candidates that distinguish the subtypes, especially for the relatively uncharacterized basal-like subtype. We also used FAIRE to investigate the set of active regulatory elements associated with the transformation of a mammary epithelial cell line to a cancerous phenotype, which included a subset of the population becoming cancer stem cells. The transformed state was achieved with only modest changes in the set of active regulatory elements (5%). The transformation can instead be attributed to relatively subtle changes in the expression of transcription factors that share a common DNA-binding site, which through both competitive and cooperative interactions at existing regulatory elements alter regulatory interactions and global expression levels. Examination of the cancer stem cells, isolated by flow-cytometry, using FAIRE revealed quite unexpectedly that these cells were in fact derived from a separate starting population. Although these findings ultimately left us with many unanswered questions, it provided us an opportunity to explore the properties regulatory components underlying the cancer stem cell phenotype. Together, these findings indicate that FAIRE will be a powerful tool for discovery of the molecular characteristics underlying cancer and that FAIRE holds promise as a clinical diagnostic tool.
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  • In Copyright
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  • "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biology."
Advisor
  • Lieb, Jason D.
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  • Chapel Hill, NC
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  • Open access
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