O papel modulador do gene AIRE - capes

MANUSCRITOAIRE on ectopic PTA gene expression is not that of a simple on/off operon;its effect on most genes is only partial, and a number of PTAs are notaffected by AIRE (Mathis and Benoist, 2007).In addition the wideness of its transcriptional impact weaken the notion ofa classical site-specific control element, as it is not easy to envision thatAIRE-binding site occurs in thousands of promoters of such disparatestructure (Mathis and Benoist, 2007).These considerations may suggest an indirect mode of action for Aire.Here, we hypothesize that PTA genes in mTECs establish networks,reflecting the existence of a feedback-like control of the transcriptionallevels. Aire in this case plays a role as upstream modulator.Material and MethodsMedullary thymic epithelial cell lineMedullary 3.10 TEC line was kindly provided by Willem van Ewijk(Rotherdam University, The Netherlands) to one of us (W.S.). This cell linewas established from C57BL/6 mice and medullary phenotype wasdetermined by immunostaining with Th-4 antibody. They was furtherevaluated in one of our laboratory (W. Savino) using a panel of anticytokeratinmonoclonal antibodies, which confirmed the original distinctmedullary phenotype. Cells were cultured in 10% fetal bovine serumsupplementedRPMI 1640 medium at 37 o C in a 5% CO 2 atmosphere.Silencing Aire gene transcriptWe used the TriFecta TM (IDT, Integrated DNA Technologies, USA) anti-Aire siRNA sequence(GGAUUCUCUUUAAGGACUACAAUCTAGAUUGUAGUCCUUAAAGAGAAUCCUC) to silencing Aire mRNA. Confluent cultures of 3.10 mTEC cell line208