The solution structure of the first PHD finger of autoimmune regulator in complex with non-modified histone H3 tail reveals the antagonistic role of H3R2 methylation

Abstract

Plant homeodomain (PHD) fingers are often present in chromatin-binding proteins and have been shown to bind histone H3 N-terminal tails. Mutations in the autoimmune regulator (AIRE) protein, which harbours two PHD fingers, cause a rare monogenic disease, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). AIRE activates the expression of tissue-specific antigens by directly binding through its first PHD finger (AIRE-PHD1) to histone H3 tails non-methylated at K4 (H3K4me0). Here, we present the solution structure of AIRE-PHD1 in complex with H3K4me0 peptide and show that AIRE-PHD1 is a highly specialized non-modified histone H3 tail reader, as post-translational modifications of the first 10 histone H3 residues reduce binding affinity. In particular, H3R2 dimethylation abrogates AIRE-PHD1 binding in vitro and reduces the in vivo activation of AIRE target genes in HEK293 cells. The observed antagonism by R2 methylation on AIRE-PHD1 binding is unique among the H3K4me0 histone readers and represents the first case of epigenetic negative cross-talk between non-methylated H3K4 and methylated H3R2. Collectively, our results point to a very specific histone code responsible for non-modified H3 tail recognition by AIRE-PHD1 and describe at atomic level one crucial step in the molecular mechanism responsible for antigen expression in the thymus.

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

“…It should also be kept in mind that these were in vitro determinations entailing an isolated recombinant Aire-PHD and a synthetic H3 amino-terminal peptide. Residual binding of the D299A mutant to H3-tails is now not so surprising, given recently published 3D solution structures of WT Aire-PHD with the H3-tail, demonstrating additional contributions by other residues, in particular an important interaction between murine D314A and H3R2, with functional consequences in both chromatin binding and PTA expression levels (5,6).…”

“…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 transcripts (at least the few tested) in cultured cell models (3,4,7).…”

Structure of human Sp140 PHD finger: an atypical fold interacting with Pin1

“…S5A). Further NMR titrations of 15 N Sp140-PHD with other H3 peptides bearing different epigenetic marks, such as H3K4me3, H3R2me2a (asymmetric di-methylation of R2) and H3K9ac, or with unmodified peptides corresponding to H3 (17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29) or H4 (1-10) did not show any binding (Fig. S6).…”

“…AIRE-PHD1; Fig. S7A) [25] is absent or partially covered by Trp728 in Sp140-PHD trans and cis conformers, respectively (Fig. S7B,C).…”

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

“…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).…”