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...
Nonalcoholic fatty liver disease (NAFLD) is a common disorder that affects millions of people around the world. People with fatty liver disease alone are usually asymptomatic until a second insult to the liver, such as oxidative stress, triggers more severe steatosis, inflammation and fibrosis resulting in nonalcoholic steatohepatitis (NASH). The additional stress and damage to the hepatocytes results in the release of signals, such as hedgehog (Hh), to induce the wound-healing response. Data from animal models and human NASH have demonstrated Hh pathway activation correlates with the severity of liver disease. (1). In addition, the transcriptional coactivator, Taz/Wwtr1, was shown to be increased in hepatocytes from human and mouse NASH livers. (2). Based on these findings we performed DGE and qRT-PCR analysis on mice fed a high fat, fructose, and cholesterol (AMLN) diet for 24 weeks and found elevated mRNA expression of Taz in the livers compared to the lean mice. We treated C57B/6 mice fed the AMLN diet for 24 weeks with ASOs targeting Taz RNA. After 12 weeks of Taz ASO treatment, we observed a 95% knockdown in Taz mRNA expression in the liver compared to the control ASO group. This was accompanied by a 38% loss in whole-body fat mass (EchoMRI) and a 69% decrease in epidydimal white adipose tissue weight. Plasma analysis demonstrated a significant decrease in glucose and cholesterol compared to the control ASO levels. In addition, we observed reductions in liver triglyceride content resulting in levels similar to a lean mouse. No improvements in liver fibrosis were observed with Taz ASO treatment. These positive effects on obesity, plasma cholesterol, and liver steatosis suggest that Taz is a potential therapeutic target for preventing the progression of NASH into a more dangerous and irreversible liver disease. Disclosure R. Peralta: None. A.K. Low: Employee; Self; Ionis Pharmaceuticals, Inc.. Other Relationship; Spouse/Partner; Abbott. J. Schmidt: Employee; Self; Ionis Pharmaceuticals, Inc.. Employee; Spouse/Partner; Ionis Pharmaceuticals, Inc.. A. Ferng: None. S. Murray: Employee; Self; Ionis Pharmaceuticals, Inc. S. Guo: Employee; Self; Ionis Pharmaceuticals, Inc..
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