Aire unleashes stalled RNA polymerase to induce ectopic gene expression in thymic epithelial cells.
Aire is a transcriptional regulator that induces expression of peripheral tissue antigens (PTA) in thymic medullary epithelial cells (MECs), driving immunological self-tolerance in differentiating T cells. To elucidate its mechanistic pathways, we examined its transcriptional impact in MECs in vivo by microarray analysis with mRNA-spanning probes. This analysis revealed initiation of Aire-activated genes to be comparable in Aire-deficient and wild-type MECs, but with a block to elongation after 50-100 bp in the absence of Aire, suggesting activation by release of stalled polymerases by Aire. In contrast, patterns of activation by transcription factors such as Klf4 were consistent with regulation of initiation. Mapping of Aire and RNA polymerase-II (Pol-II) by ChIP and high-throughput sequencing (ChIP-seq) revealed that Aire bound all Pol-II-rich transcriptional start sites (TSS), irrespective of its eventual effect. However, the genes it preferentially activated were characterized by a relative surfeit of stalled polymerases at the TSS, which resolved once Aire was introduced into cells. Thus, transcript mapping and ChIP-seq data indicate that Aire activates ectopic transcription not through specific recognition of PTA gene promoters but by releasing stalled polymerases.
Division of Immunology, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115, USA.
Matthieu
M
Giraud
Hideyuki
H
Yoshida
Jakub
J
Abramson
Peter B
PB
Rahl
Richard A
RA
Young
Diane
D
Mathis
Christophe
C
Benoist
D000818:Animals; D002460:Cell Line; D047369:Chromatin Immunoprecipitation; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000276:immunology*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D000090062:Kruppel-Like Factor 4; D051379:Mice; D008810:Mice, Inbred C57BL; D046228:Microarray Analysis; D017634:Self Tolerance / Q000276:immunology*; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D013950:Thymus Gland / Q000166:cytology*; D014157:Transcription Factors / Q000378:metabolism*; D000096927:AIRE Protein
D012321:DNA-Directed RNA Polymerases; D000090062:Kruppel-Like Factor 4; D014157:Transcription Factors; D000096927:AIRE Protein; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse