Summary During development, the precise relationships between transcription and chromatin modifications often remain unclear. We use the X chromosome inactivation (XCI) paradigm to explore the implication of chromatin changes in gene silencing. Using female mouse embryonic stem cells, we initiate XCI by inducing Xist and then monitor the temporal changes in transcription and chromatin by allele-specific profiling. This reveals histone deacetylation and H2AK119 ubiquitination as the earliest chromatin alterations during XCI. We show that HDAC3 is pre-bound on the X chromosome and that, upon Xist coating, its activity is required for efficient gene silencing. We also reveal that first PRC1-associated H2AK119Ub and then PRC2-associated H3K27me3 accumulate initially at large intergenic domains that can then spread into genes only in the context of histone deacetylation and gene silencing. Our results reveal the hierarchy of chromatin events during the initiation of XCI and identify key roles for chromatin in the early steps of transcriptional silencing.

Homozygous loss-of-function mutations in AIRE cause autoimmune polyglandular syndrome type 1 (APS 1), which manifests in a classic triad of hypoparathyroidism, adrenal insufficiency, and candidiasis. Interestingly, a kindred with a specific G228W AIRE variant presented with an autosomal dominant autoimmune phenotype distinct from APS 1. We utilized a novel G228W-knockin mouse model to show that this variant acted in a dominant-negative manner to cause a unique autoimmunity syndrome. In addition, the expression of a large number of Aire-regulated thymic antigens was partially inhibited in these animals, demonstrating the importance of quantitative changes in thymic antigen expression in determining organ-specific autoimmunity. Furthermore, the dominant-negative effect of the G228W variant was exerted through recruitment of WT Aire away from active sites of transcription in the nucleus of medullary thymic epithelial cells in vivo. Together, these results may demonstrate a mechanism by which autoimmune predisposition to phenotypes distinct from APS 1 can be mediated in a dominant-negative fashion by Aire.

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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...

Autoimmune regulator (AIRE) is a transcription factor that induces the expression of a large subset of otherwise strictly tissue restricted antigens in medullary thymic epithelial cells, thereby enabling their presentation to developing T cells for negative selection. Mutations in AIRE lead to autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a rare monogenetic disease. Although it has been reported that AIRE interacts with proteins involved in nuclear transport, DNA-damage response, chromatin remodeling, transcription and pre-mRNA-splicing, the precise mechanism of AIRE-induced tissue restricted antigen expression has remained elusive. In this study, we investigated an APECED patient mutation that causes the loss of the extreme C-terminus of AIRE and found that this mutant protein is transcriptionaly inactive. When tethered heterologously to DNA, this domain could stimulate transcription and splicing by itself. Moreover, the loss of this C-terminus disrupted interactions with the positive transcription elongation factor b (P-TEFb). Via P-TEFb, AIRE increased levels of RNA polymerase II on and enhanced pre-mRNA splicing of heterologous and endogenous target genes. Indeed, the inhibition of CDK9, the kinase subunit of P-TEFb, inhibited AIRE-induced pre-mRNA splicing of these genes. Thus, AIRE requires P-TEFb to activate transcription elongation and co-transcriptional processing of target genes.

The autoimmune regulator is a critical transcription factor for generating central tolerance in the thymus. Recent studies have revealed how the autoimmune regulator targets many otherwise tissue-restricted Ag genes to enable negative selection of autoreactive T cells.

The autoimmune regulator (AIRE) is a transcription factor which is expressed in medullary thymic epithelial cells. It directs the expression of otherwise tissue-specific antigens, which leads to the elimination of autoreactive T cells during development. AIRE is modified post-translationally by phosphorylation and ubiquitylation. In this report we connected these modifications. AIRE, which is phosphorylated on two specific residues near its N terminus, then binds to the F-box protein 3 (FBXO3) E3 ubiquitin ligase. In turn, this SCF FBXO3 (SKP1-CUL1-F box) complex ubiquitylates AIRE, increases its binding to the positive transcription elongation factor b (P-TEFb), and potentiates its transcriptional activity. Because P-TEFb is required for the transition from initiation to elongation of transcription, this interaction ensures proper expression of AIRE-responsive tissue-specific antigens in the thymus.