The Autoimmune Regulator PHD finger binds non-methylated Histone H3K4 to activate gene expression

Mutations in AIRE gene cause a monogenic autoimmune disease named autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). Thus, APECED is recognized as a unique model for molecular studies of autoimmunity and immunological tolerance. AIRE is expressed in thymic medullary epithelial cells where it promotes the expression of tissue-restricted antigens. Based on these data, AIRE role in epigenetic regulation has been proposed. AIRE harbors two PHD fingers. Recently PHD finger domains were reported to preferentially bind to the trimethylated forms of lysine 4 of Histone H3 (H3K4). By the combined use of biochemical and biophysical (heteronuclear NMR spectroscopy, fluorescence spectroscopy, ITC) methods we show that AIRE selectively interacts with histone H3 through its first PHD finger domain. AIRE-PHD1 discriminates between different degrees of histone H3 lysine 4 (H3K4) methylation and preferentially binds non-methylated H3K4 (KD ~4 uM). Molecular dynamics calculations of the AIRE-PHD1 interaction with unmodified H3K4 strongly support the experimental NMR data: the peptide binds on the surface of AIRE-PHD1 creating several stable hydrogen bonds with the first beta-strand. The interaction is further stabilized by a salt bridge between the side-chains of K4 and D297, and between R2 and D312. This is further confirmed by site-directed mutagenesis which shows the importance of the ionic contacts between R2-D312 and K4-D297 in complex formation. In vivo AIRE binds and activates promoters (involucrin, insulin) containing non-methylated H3K4 in HEK293 cells. We propose that AIRE-PHD1 is a prototype of a new class of PHD fingers that specifically recognize non-methylated H3K4, thus providing a new link between the status of histone modifications and the regulation of tissue-restricted antigen expression in thymus.