DNA methylation landscape in mast cell differentiation and systemic mastocytosis
Methylation of cytosines in the mammalian genome represents a key epigenetic modification regulating gene transcription, and is dynamically regulated during differentiation and disease. New DNA methylation patterns are established by the de novo DNA methyltransferase enzymes DNMT3A and DNMT3B. During DNA synthesis, the global methylation landscape is copied onto the daughter DNA strand by the maintenance DNA methyltransferase DNMT1, thus warranting its heritability across mitosis, which is essential both for regulation of gene expression and maintenance of genomic stability. A recently identified family of DNA-modifying enzymes, the TET family of proteins, was shown to modify 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) and to further oxidation products. These different modifications of the methyl mark can directly lead to specific transcriptional effects, while further oxidation and deamination of the modified base can eventually lead to loss of the methyl mark and heritable reprogramming of the epigenome. Importantly, alterations of the DNA methylation landscapes as well as mutations in specific DNA-modifying enzymes (most commonly DNMT3A and TET2) are frequently observed in different types of leukemia, but the specific mechanisms by which altered DNA methylation promotes tumorigenesis remain largely unknown. The role, if any, of altered DNA methylation in myeloproliferative disorders, namely pre-neoplastic conditions associated with dysregulated production of myeloid cells, is not understood. Our own unpublished data point toward a role for DNA methylation dynamics and TET2 in systemic mastocytosis, a myeloproliferative disorder characterized by the abnormal proliferation and accumulation of aberrant mast cells. In this project, we set out to dissect the impact of perturbing the DNA methylation landscape during myeloid differentiation to mast cells, as well as its effect on mast cell proliferation and function in normal and pathologic conditions. Specifically, I propose to investigate the following Aims:Aim 1: DNA methylation landscape in the presence or absence of DNA-modifying enzymes. With the goal of carefully dissecting DNA methylation dynamics during differentiation, we will analyse the genomic landscape of 5hmC/5mC as well as the expression of TET and DNMT enzymes during differentiation of mast cells from hematopoietic progenitors. Aim 2: Effects of mutations in DNA-modifying enzymes. Since Dnmt3a and Tet2 mutations play a role in the pathogenesis of myeloproliferative disorders, including systemic mastocytosis, we will investigate the effect of such mutations in mast cell differentiation and function, both in vitro and in vivo.Aim 3: Interplay between DNA-modifying enzymes and microRNAs. We will investigate how modifications in DNA methylation modulate microRNA expression, and also how microRNAs may reciprocally modulate expression and activity of DNA-modifying enzymes, thus impacting on the DNA methylation landscape. The hypotheses tested in this project are strongly supported by preliminary data in our own laboratory as well as by the available literature. These studies will significantly expand our understanding of the mechanisms underlying fundamental processes of myeloid cell production during normal homeostasis and disease.