The goal of this project is to understand the role of histone recognition in the control of central tolerance by Aire. Aire, for autoimmune regulator, is a transcription factor that has an obligatory role in maintaining T cell tolerance to self-tissues1. In its absence, gene-targeted mice and human APECED (autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy) patients develop T cell-mediated autoimmunity, because Aire-dependent expression of tissue restricted antigens (TRAs) in the thymus is essential for deletion of autoreactive thymocytes. But how Aire functions to derepress these genes, which are normally silenced in all but a few specific cell types, is not known1. Identifying the mechanisms of Aire function would be invaluable for understanding the molecular basis of APECED disease. This effort would also reveal novel insights into gene silencing, which restricts developmental potential during cellular differentiation and is overcome during neoplastic transformation of differentiated cells. We have recently demonstrated an in vitro histone-binding activity for PHD1, one of the PHD domains of Aire. We have identified residues in PHD1 critical for this association. Furthermore, we show that histone binding is disrupted in all previously characterized APECED mutations mapping to PHD1. Therefore, we hypothesize that histone recognition by PHD1 is essential for Aire function in vivo. The proposed research plan will rigorously test this hypothesis.
In Aim 1, we will evaluate wild-type Aire and mutants'defective for in vitro binding described above, for in vivo association with histones, and for their ability to reconstitute TRA induction in a cell-based functional assay.
In Aim 2, we will generate """"""""knock-in"""""""" mice bearing mutations that disrupt histone binding and TRA induction, as determined in Aim 1 above. The goal is to evaluate these mice for the development of spontaneous autoimmunity, in order to assess the role for PHD1-mediated histone recognition in Aire-dependent central tolerance.
In this proposal we plan to evaluate one aspect of the function of a protein called Aire. Aire is a transcription factor that turns on genes necessary for eliminating autoreactive T cells. When Aire is mutated, as in human APECED patients, T cells attack self-tissues and cause autoimmunity. We have uncovered the means by which Aire can recognize its genes. We will use both cell-based and mouse models to test the hypothesis that this recognition is indeed critical for Aire to turn on its genes and prevent autoimmunity.
