The Ten-eleven translocation (Tet1/2/3) family of enzymes are epigenetic regulators of gene expression important for stem cell biology and embryonic development. Tet enzymes are dioxygenases that promote DNA demethylation by converting 5-methylcytosine (5mC) into 5-hydroxymethycytosine (5hmC) and higher oxidized derivatives. In addition to this enzymatic activity, Tet enzymes can bind chromatin modifying complexes, to regulate genes in a presumably catalytic-independent manner. Tet2 is a key member of this family. It is highly expressed in embryonic stem cells (ESCs) and controls gene expression programs necessary for stem cell lineage specification. Tet2 is also frequently mutated in hematological malignancies and has been implicated in neurodegenerative diseases. While the catalytic functions of Tet2 have been well studied, its non-catalytic roles remain poorly defined. In this proposal, we seek to establish the significance of the catalytic dependent and independent functions of Tet2 in ESC gene regulation and lineage commitment. We hypothesize that Tet2, in addition to regulating genes through its DNA demethylase activity, can also modulate genes in a non-catalytic fashion by recruiting histone modifiers to the chromatin, and this dual mode of gene regulation is essential for proper ESC differentiation along the neural and hematopoietic lineages. To test this hypothesis, I have generated Tet2 catalytic mutant (Tet2m/m) and knock-out (Tet2?/?) ESCs, which I will use as a platform to: (1) identify the catalytic and non-catalytic direct target genes of Tet2 in ESCs by integrating changes in gene expression with Tet2 genomic occupancy, (2) establish Tet2-mediated activating and repressing mechanisms of gene regulation involving interactions with histone modifiers OGT and HDAC2, and finally (3) define the biological significance of Tet2 enzymatic and non-enzymatic functions in ESC differentiation and lineage commitment along the neural and hematopoietic lineages. Findings from these experiments will elucidate novel epigenetic mechanisms of gene regulation in ESCs involving Tet2 catalytic and non-catalytic functions. They will enhance our understanding of stem cell biology and development and can have implications in hematological malignancies where Tet2 is affected. Under the combined mentorship of Drs. Meelad Dawlaty and Bernice Morrow, I will successfully execute the proposed research and training plans. This will allow me to contribute greatly to the fields of epigenetics and stem cell biology and develop the necessary research, professional and interpersonal skills to become and independent physician-scientist investigator.
Given the promise of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in regenerative medicine, investigating their biology is of paramount significance to human health. This proposal investigates the role of Tet2, an important epigenetic modifier that regulates ESC biology and is also frequently mutated in hematological malignancies. Findings from these studies will enhance our understanding of gene regulatory mechanisms in stem cell biology and could lead to identification of new targets for stem cell therapuetics and treatment of diseases such as cancer.
