PROJECT ABSTRACT My lab is focused on the epigenetic and transcriptional regulation of lineage specification. Perturbations in the process of lineage specification can result in human disorders, such as malignancies and developmental syndromes. During the process of lineage specification, multipotent precursor cells give rise to progeny cells with specialized, characteristic patterns of gene expression. Lineage-specifying transcription factors drive cell- specific gene expression programs, but their access to the DNA is finely tuned by epigenetic machinery that regulate DNA methylation and histone modifications. Importantly, lineage-specifying transcription factors cannot bind to methylated cytosines in DNA. A fundamental step in the establishment of cell fate is the unmasking of specific transcription factor binding sites by targeted removal of methylated cytosines. This process is tightly regulated by the Ten Eleven Translocation (TET) family of proteins that share a catalytic domain and can oxidize 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC) and other oxidized cytosines. Each modified cytosine is a stable epigenetic mark that can be preferentially recognized by transcription factors. In a given cell type there can be simultaneous expression of at least two of the three TET family members. Though the function of each protein is obscure, our previous research revealed a fundamental role of TET proteins in fine-tuning the expression of lineage specification factors. We hypothesize that TET proteins act in concert with largely unknown, cell-specific, pioneer transcription factors. In progenitor cells, these pioneer factors anchor TET proteins to specific loci, still inaccessible to lineage-specifying transcription factors. Then TET proteins initiate the process of 5mC oxidization, allowing for orchestrated recruitment of lineage specifying transcription factors. The overarching mission of our research is to decipher the TET mediated mechanisms that regulate cell lineage choice and specification. We will utilize genomic, genetic and biochemical approaches to investigate changes in modified cytosine (5hmC), chromatin accessibility, and gene expression to: 1) dissect the shared versus the distinct functions of TET proteins; 2) determine whether TET proteins function through canonical, catalytic- dependent activities or have additional, unexpected, catalytic-independent mechanisms; 3) identify the factors that can interact with TET proteins in sequential snapshots of lineage specification. Upon completion of our work, we will elucidate the precise and multifaceted mechanisms by which TET proteins influence lineage specification and affect human disorders.