Unmodified Histone H3K4 and DNA-Dependent Protein Kinase Recruit Autoimmune Regulator to Target Genes

Abstract

c Autoimmune regulator (AIRE) directs the expression of otherwise tissue-restricted antigens (TRAs) in medullary thymic epithelial cells, allowing their presentation to developing T cells, which leads to central tolerance. We addressed the conundrum of how AIRE is recruited to these otherwise silent genes in cells. Our studies confirmed that interactions between AIRE and the unmodified histone H3K4 (H3K4me0) are important for targeting AIRE to the mouse insulin promoter in chromatin. By replacing its H3K4me0-binding module with one that binds to the methylated H3K4me3, we redirected the mutant AIRE.ING protein to an actively transcribed gene. Nevertheless, the mutant AIRE D297A protein, which could not bind to H3K4me0, still activated the human insulin promoter on an episomal plasmid target. This targeting was due to DNA-dependent protein kinase (DNA-PK). Thus, in cells that lacked the catalytic subunit of DNA-PK (DNA-PKcs), the assembly and activity of AIRE on DNA, whether in chromatin or on episomal plasmids, was abrogated. However, by the heterologous tethering of AIRE to DNA, we could restore its activity on a plasmid target in DNA-PKcs-negative cells. Importantly, mutations in the putative DNA-binding residues in its SAND domain had no effect on the transcriptional effects of AIRE. Thus, AIRE is recruited to TRA genes in chromatin via cooperative interactions with H3K4me0 and DNA-PK. The negative selection of developing T cells in the thymus is an essential element of the mammalian immune system. In medullary thymic epithelial cells (mTECs), the protein that governs central tolerance is the autoimmune regulator (AIRE) (3,26,29,39). Mutations in AIRE lead to a rare genetic autoimmunity called the autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), which affects mostly endocrine and exocrine organs, such as salivary, adrenal, thyroid, and parathyroid glands, as well as the ␤ cells of the pancreas (1, 13, 33). In mTECs, AIRE activates the expression of otherwise silent tissue-restricted antigen (TRA) genes, whose peptides are presented on major histocompatibility complex class II (MHC-II) determinants to thymocytes so that autoreactive T cells are eliminated (3,26,29,39).AIRE is a transcription factor (TF) of 57 kDa and contains 545 residues (13, 33). It forms oligomers that migrate as dimers, tetramers, and higher order aggregates, which can exceed 670 kDa (17). Thus, 12 or more AIRE proteins could be present in these large complexes. However, the stoichiometry of the transcriptionally active complex is unknown. From its N terminus, AIRE contains a homogenous staining region (HSR); an Sp100, Aire-1, NucP41/75, and DEAF-1 (SAND) domain, which has been suggested to bind DNA; two plant homeodomains (PHD1 and PHD2), of which PHD1 binds unmodified histone H3K4 (H3K4me0) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs); and finally a transcriptional activation domain (TAD), which binds the positive transcription elongation factor b (P-TEFb) at its C terminus (24,34,40,47). H...

Ovarian autoimmune disease: clinical concepts and animal models

“…[78][79][80] These PHD domains associate with histone 3 at unmethylated lysine 4 residues (H3K4me0), which are typically within transcriptionally inactive chromatin regions, and in combination with DNA-dependent protein kinase, appear to promote subsequent transcription. [81][82][83] Consistent with this model of transcriptional regulation, AIREregulated genes are chromosomally clustered and stochastically expressed. 68,[84][85][86] Additionally, immunochemical localization suggests that AIRE associates with the nuclear matrix in a distinctive speckling pattern (Figure 2).…”

Brd4 bridges the transcriptional regulators, Aire and P-TEFb, to promote elongation of peripheral-tissue antigen transcripts in thymic stromal cells

“…Aire is also known to be phosphorylated; specifically, T69 and S157 are targeted by DNA-dependent protein-kinase (DNA-PK) (31), which has been shown by multiple approaches to be a major Aire-interacting protein in both MECs and model cell lines (31)(32)(33). Co-IP experiments using Aire/Flagtransfected 4D6 cells pretreated or not with the specific DNA-PK inhibitor, NU7441, revealed Aire's interaction with Brd4 to depend on a phosphorylation event mediated by this kinase (Fig.…”

FBXO3 Protein Promotes Ubiquitylation and Transcriptional Activity of AIRE (Autoimmune Regulator)

“…Their presentation to developing T cells allows self-reacting cells to be eliminated. In these cells, AIRE is recruited to the stalled RNA polymerase II (RNAPII) on TSA promoters by the unmodified histone H3K4 and DNA-dependent protein kinase (DNA-PK) (7)(8)(9)(10). DNA-PK also phosphorylates AIRE for increased transcriptional activity (11).…”