ORCID
0000-0002-6087-1962
Date of Award
5-3-2024
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Acute myeloid leukemia (AML) is a genetically and phenotypically heterogeneous disease. Recurrent mutations in genes involved in epigenetic pathways are common in AML and are thought to contribute to this variability by disrupting epigenetic patterns relative to normal hematopoietic cells. The most well-studied epigenetic modification in AML is DNA methylation. Although methods for studying DNA methylation have improved significantly in the past decade, the landscape of methylation changes in AML and how they relate to chromatin architecture and gene regulation is still not fully understood. This thesis is focused on the DNA methylation phenotype of IDH1 or IDH2 mutations in AML cells and the functional consequences of these changes. IDH1 or IDH2 mutations occur in approximately 20% of AML patients and are associated with altered DNA methylation patterns. The IDH1 and IDH2 genes encode metabolic enzymes that are not normally involved in DNA methylation. However, the mutant forms of IDH1 and IDH2 in AML produce 2-hydroxyglutarate (2HG) that inhibits the TET family of enzymes, which act to remove methylated cytosines on DNA through successive oxidation reactions. Hence, the net effect of IDH mutations is an increase in DNA methylation by inhibiting its removal. Although the specific consequences of this hypermethylation phenotype have been difficult to define, in vivo mouse models have established that IDH mutations contribute to AML development. We used a comprehensive set of whole-genome bisulfite sequencing data from primary AML to characterize AML-associated DNA methylation phenotypes and identify IDH mutation-specific methylation changes in primary AML samples. Such an approach had not yet been taken to understand the direct consequences of IDH mutations, and our study identified a unique hypermethylation signature that is enriched for active enhancer regions in IDH-mutant AML. The differentially methylated regions (DMRs) from this analysis were characterized by active methylation and demethylation turnover, evidenced by an increase in TET-mediated 5-hydroxymethylation, which may account for the unique susceptibility of enhancers to become hypermethylated in the presence of IDH mutations. We then used high-resolution chromatin conformation data to link these IDHmut-specific hypermethylated enhancer DMRs with their cognate gene(s), which we found to be highly expressed in hematopoietic cells. The interactions often coincided with CTCF binding sites that orchestrated chromatin loop formation and formed insulating TAD boundaries. We observed some evidence of disruption of loop formation and boundary insulation in IDH mutant samples compared with CD34+ normal hematopoietic cells, which correlated with decreased target gene expression in a subset of interactions. These results suggest that hypermethylation accumulates at regions that are important for genome organization and in certain cases may affect gene expression in favor of leukemia development. Mechanistic studies of candidate loci were performed to better define the nature of enhancer DMR regulatory interactions. Results from targeted deletion of the DMR in a well-characterized MYC enhancer were consistent with our hypothesis of inherent regulatory activity associated with DMR regions, demonstrating a trend towards decreased MYC expression in DMR KO cell lines compared with WT cells.
Language
English (en)
Chair and Committee
David Spencer
Recommended Citation
Wilson, Elisabeth, "Integrating DNA Methylation and 3D-genome Architecture to Identify Functional Regulatory Sequences in IDH-mutant AML" (2024). Arts & Sciences Electronic Theses and Dissertations. 3072.
https://openscholarship.wustl.edu/art_sci_etds/3072