Abstract
Autoimmune regulator (AIRE) is responsible for the development of organ-specific autoimmune disease in a monogenic fashion. Rare and low levels of tissue expression together with the lack of AIRE-expressing cell lines have hampered a detailed analysis of the molecular dynamics of AIRE. Here we have established cell lines stably transfected with AIRE and studied the regulatory mechanisms for its subcellular expression. We found that nuclear body (NB) formation by AIRE was dependent on the cell cycle. Biochemical fractionation revealed that a significant proportion of AIRE is associated with the nuclear matrix, which directs the functional domains of chromatin to provide sites for gene regulation. Upon proteasome inhibition, AIRE NBs were increased with concomitant reduced expression in the cytoplasm, suggesting that subcellular targeting of AIRE is regulated by a ubiquitin-proteasome pathway. We also found that AIRE NBs compete for cAMP-response element-binding protein-binding protein/p300, a common coactivator of transcription, with the promyelocytic leukemia gene product. These results suggest that the transcriptional regulating activities of AIRE within a cell are controlled and organized in a spatiotemporal manner.
Highlights
Breakdown of self-tolerance is considered to be the key event for the development of autoimmune diseases [1]
Establishment of Stable Transfectants Expressing autoimmune regulator (AIRE)—It has been demonstrated that AIRE is predominantly expressed in the Medullary thymic epithelial cell (mTEC) with the characteristic morphology of nuclear body (NB) [11,12,13]
The low levels of expression together with the lack of established cell lines constitutively expressing AIRE have hampered a detailed analysis of the molecular dynamics of AIRE
Summary
Construction of the Fusion Gene for AIRE cDNA with Marker Genes— Human AIRE cDNA was amplified by PCR from Marathon-Ready human thymus cDNA (BD Biosciences, San Jose, CA). After isolation of the cytoplasmic protein, nuclear pellets were resuspended in 50 l of cytoskeletal (CSK) buffer (10 mM PIPES (pH 6.8), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, 1 mM dithiothreitol, and protease inhibitors) containing 100 units of RNase-free DNase I (Roche Applied Science) and incubated for 50 min at 30 °C. Immunoprecipitation with anti-Myc mAb (clone 9E10) and Western blot analysis with anti-HA polyclonal Ab, both from Santa Cruz Biotechnology, were performed as described previously [16, 17]. Samples were resolved by SDS-PAGE on a 6% gel and subjected to Western blot analysis with a mouse mAb to Ub (clone 1B3; MBL, Nagoya, Japan) and horseradish peroxidase-conjugated rabbit polyclonal Ab to mouse immunoglobulin (Promega).
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