Elucidating the molecular mechanism of TET2 function in hematopoiesis

DNA modifications are epigenetic marks deposited throughout the mammalian genome and play an important role in gene regulation in normal and malignant biological processes. Biological DNA modifications are introduced by DNA methyltransferases (DNMT) and dioxygenases of ten- eleven translocation (TET) family. DNMT proteins can convert cytosine into 5-methylcytosine. TET proteins catalyze the oxidation of 5mC to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. These oxidized forms of cytosine stay in the DNA or result in DNA demethylation. TET2, a member of TET family proteins, is one of the most frequently mutated genes in hematological malignancies. While TET2 has roles in normal hematopoiesis, including stem cell self-renewal and specific lineage commitment, the exact molecular mechanism of its function still remains unknown. The aims of the project are to identify the role of TET2 in regulating hematopoietic gene expression and to identify the molecular partners of TET2 in the hematopoietic-associated context, thus to arrive at the mechanism of action of TET2 in normal hematopoiesis. To study the Tet2 function in a hematopoietic context, I chose the mouse hematopoietic progenitor cell line, HPC-7, as a model system. I constructed the Tet2-knock-out (KO) HPC-7 cell and characterized the cell differentiation phenotype caused by Tet2 depletion. To understand the role of Tet2 in gene regulation, I firstly identified genes affected by Tet2 depletion. Then I mapped the Tet2 binding profile in HPC-7 and discovered genes directly targeted by Tet2. Finally, I assessed the contribution of DNA demethylation in Tet2-mediated gene regulation. The results revealed that Tet2 deficiency caused a block of cell differentiation in the Colony-forming-unit assay. Genes involved in hematopoiesis were differentially expressed upon Tet2 deficiency. Surprisingly, Tet2 binding is associated with both gene activation and gene repression. Tet2 binds to the hematopoietic-enhancers together with many hematopoietic transcription factors, and leads to DNA demethylation at enhancers, thus activates the expression of essential hematopoietic genes, such as csf1R, Cebpa, Cebpe. The mechanism by which Tet2 negatively regulates its targeted gene expression needs further investigation. To identify Tet2 molecular partners in the hematopoietic-associated context, I mapped the TET2 interactome in HPC-7 cells by immunoprecipitation coupled to mass spectrometry. Our data suggests that polycomb repressive complex 2 (PRC2) physically interacts with Tet2 via the C- terminus of Tet2 protein. The functional importance of Tet2-PRC2 interaction in gene regulation and cell differentiation remain further explored. Taken together, this study systematically investigated the Tet2-mediated gene regulation and identified new Tet2 binding partner, PRC2 complex, suggesting a novel mechanism of Tet2 function in hematopoiesis.