The epigenetic mark of DNA methylation is established by DNMT (DNA methyltransferase) enzymes and has been shown to correlate with transcriptional states and influence cell identity and tumorigenesis in mammalian cells. The recent discovery that TET (Ten-Eleven-Translocation) enzymes produce 5-hydromethylcytosine (5hmC), 5-formylcytosine (5fC), 5-carboxycytosine (5caC) and mediate active DNA demethylation in the genome has opened a new avenue to understand how DNA methylation dynamics affect transcriptional programs1. Mutations in TET2 and DNMT3A are frequently found (~10-50% of patients) in a wide range of blood diseases, including Acute Myeloid Leukemia (AML) and Myelodysplastic syndrome (MDS). However, the downstream events that cause hematopoietic stem cell to expand and transform following the occurrence of these mutations are currently unknown.
Genome-wide analysis of DNA methylation patterns has demonstrated that the products of TET-mediated cytosine oxidation (5hmC, 5fC and 5caC) accumulate at gene regulatory elements, especially at promoter-distal enhancer elements. In addition, TET2 binds directly to enhancers and genetic disruption of TET2 in hematopoietic cells results in enhancer DNA hypermethylation2. Likewise, deletion of DNMT3A has also been shown to affect enhancer DNA methylation, although with the opposite result (hypomethylation), and to promote cancerous transformation3. Thus, the growth advantage observed in TET2- and DNMT3A-mutated cells may be causally related to aberrant DNA methylation at poised and active enhancers that, in turn, leads to deregulation of genes and pathways associated with hematopoietic stem cell differentiation and self-renewal.
This PhD project aims at dissecting the global role of aberrant DNA methylation at hematopoietic enhancers. Disruption of the DNA methylation machinery will be performed using CRISPR-based gene editing in existing and novel models of native and malignant hematopoiesis. We will perform quantitative analysis of different aspects of enhancer function by genome-wide approaches (RNA-seq, ATAC-seq, and Capture BS-seq) to systematically identify DNA methylation-sensitive enhancers and discover aspects of chromatin and gene expression influenced by aberrant DNA methylation states. Computational analysis and multi-layered data integration will be performed using novel and established bioinformatics tools. At the completion of this project, the PhD candidate will have obtained a strong skill set in CRISPR-based gene editing, epigenetics and computational biology.
1. Rasmussen, K. D. & Helin, K. Role of TET enzymes in DNA methylation, development, and cancer. Genes Dev. 30, 733–750 (2016).
2. Rasmussen, K. D. et al. Loss of TET2 in hematopoietic cells leads to DNA hypermethylation of active enhancers and induction of leukemogenesis. Genes Dev. 29, 910–922 (2015).
3. Leukemias, F. et al. DNMT3A Loss Drives Enhancer Hypomethylation in Article DNMT3A Loss Drives Enhancer Hypomethylation in FLT3-ITD-Associated Leukemias. Cancer Cell 922–934 (2016). doi:10.1016/j.ccell.2016.05.003