Epigenetic regulators often lie downstream of signaling pathways that culminate in post-translational modifications, which affect their activity and ensure appropriate transcriptional responses to developmental and environmental cues. Our preliminary studies indicate that an interaction between epigenetic regulator, TET3, and an X-linked, post-translational modification enzyme, OGT, may be central in detecting X chromosome dosage and poising cells for X-inactivation. We propose to investigate the role of the TET3-OGT interaction in regulation of X chromosome inactivation, to obtain molecular insight into how cells count X chromosomes.
How cells are able to determine X chromosome number and use this information to trigger X chromosome inactivation is one of the major unsolved mysteries in the mammalian dosage compensation field. Our preliminary data suggest that a complex containing X-linked and autosomal proteins exhibits different activity in XY male and XX female cells at a developmental stage that precedes X-inactivation. This complex regulates DNA methylation, which is central in the regulation of a key player in X-chromosome silencing, the Xist gene. Our data suggest that an XX-specific complex containing the X-linked protein, OGT, and the autosomal protein, TET3, determines the low levels of DNA methylation that allow Xist up regulation in XX cells. OGT is a post-translational modification enzyme that adds a single O-linked sugar to nuclear, cytoplasmic, and mitochondrial proteins. OGT interacts with and modifies TET3, a dioxygenase that converts 5-methylcytosine to 5-hydroxymethylcytosine and higher oxidized states. These oxidation states of 5-methylcytosine are not only transient intermediates in DNA demethylation, but they can also exist as stable epigenetic marks that regulate gene expression. We propose to determine how the increased dose of X-linked OGT prior to X- chromosome silencing results in the production of an XX-specific complex, and how this complex functions to control cytosine modifications and Xist expression. These studies will provide molecular insight into a key issue in mammalian dosage compensation.
