Global relevance of Aire binding to hypomethylated lysine-4 of histone-3

Abstract

Aire promotes the ectopic expression of a repertoire of peripheraltissue antigens (PTAs) in thymic medullary epithelial cells (MECs) to mediate deletional tolerance and thereby prevent autoimmunity. Binding of hypomethylated histone 3 (H3)-tails by Aire's plant homeodomain (PHD) finger is essential for Aire function in cultured cell models, prompting speculation that Aire-PHD:H3-tail interactions underlie targeting of Aire to weakly transcribed loci. To evaluate the role of Aire's PHD finger in MECs on a global scale in vivo, we complemented Aire-deficient mice with a mutant of Aire that inhibits its binding to hypomethylated H3K4 residues. Although the range of Aire-targeted genes was largely unaffected in these mice, the D299A mutation caused a global dampening of Aire's transcriptional impact, resulting in an autoimmune disease similar in profile to that of their Aire-deficient counterparts. To test whether a low H3K4 methylation state is sufficient for Aire targeting, we overexpressed an H3K4-specific demethylase in an Aire-dependent cultured cell system, and determined its capacity to extend Aire's transcriptional footprint. The range and magnitude of Aire-regulated genes was largely unaffected, the only genes additionally induced by Aire in this context being those already accessed for repression. In short, Aire's H3-binding module is necessary for Aire-mediated regulation of gene expression and central tolerance induction, but this influence is unlikely to reflect a targeting mechanism solely based on the recognition of hypomethylated H3K4 residues.T o guard against pathogens vast in range and number, the adaptive immune system maximizes antigen-receptor diversity by using a set of random gene rearrangement processes. As reactivity to self is an unavoidable outcome of these events, mechanisms to impose immunological tolerance are needed to prevent autoimmune pathology. For αβ T cells, tolerance relies, in part, on thymic MECs, which ectopically express thousands of PTAs, the presentation of which purges differentiating thymocytes of corresponding specificities (1). Mutations in the transcriptional regulator Aire compromise this ectopic expression, resulting in multiorgan autoimmune disease both in humans and in mice (2).Precisely how Aire regulates PTA expression is under active investigation. Of late, much attention has focused on a PHD finger located near the center of the amino acid sequence. It was recently demonstrated that this structural element is a histone-binding module, specific for H3-tails impoverished in posttranslational modifications (3, 4). The Aire PHD forms an anti-parallel β-strand-like conformation in conjunction with the amino terminus of H3, anchored by electrostatic interactions between its aspartate/ glutamate residues and unmodified H3R2, H3T3, H3K4 and H3R8 amino acids (5, 6). Disruption of certain of these interactions either by posttranslational modification of an H3 residue or by mutation of an Aire residue compromised both Aire's binding to chromatin and its induction of PTA...

Aire’s plant homeodomain(PHD)-2 is critical for induction of immunological tolerance

“…This effect is similar to that reported for a debilitating point mutation in PHD1 (19), and both alterations led to a multiorgan autoimmune disease essentially indistinguishable from that of Aire-KO mice. At present, we have no explanation for why a small subset of genes was notably less affected by the ΔPHD2 than the Aire-null mutation.…”

“…However, it may be relevant that several of the genes highlighted as being less responsive to the former than the latter are members of multigene families (e.g., Mup) whose members are located together on the chromosome. Interestingly, some of the same loci were previously found to be less affected by a debilitating PHD1 point mutation than the Aire-null mutation (19).…”

“…NOD/Lt J (Aire-WT) mice were originally purchased from the Jackson Laboratory. Aire-KO and iA-WT mice on the NOD genetic background have been described (19,27). An Aire cDNA carrying PHD2-domain deletion (amino acids 434-475) was constructed by QuikChangeII XL (Stratagene) and introduced into the ClaI site of the TOA construct (19).…”

“…To address the function of Aire's PHD2 in vivo, we used a described system (26) to generate lines of transgenic mice expressing either WT Aire (iA-WT) or PHD2-deleted Aire (iA-ΔPHD2) quasi-specifically in thymic MECs, on an Aireknockout (Aire-KO) nonobese diabetic (NOD) background. Mice generated by using this system faithfully recapitulate the critical features of Aire control of immunological tolerance and have been used previously to examine the role of PHD1 in Aire's activities (19), as well as temporal aspects of Aire's impact on tolerance (27).…”

“…1A), which generally function in protein:protein interactions, notably as histone "readers" or as docking domains involved in stabilization of multiprotein complexes in various stages of transcription (14). The more amino-terminal PHD1 is a functionally critical region of Aire, acting as a reader of histone-3 molecules unmethylated at the lysine-4 residue (H3K4me0) and thereby promoting targeting to transcriptionally dormant genes (15)(16)(17)(18)(19)(20). PHD1 has also been reported to be a vital link in Aire's interaction with DNA-PKcs (20) and to harbor E3 ubiquitin-ligase activity essential for transcriptional induction (21), although the NMR solution structure and subsequent functional assessments did not support this latter role (22).…”