title: An RNAi screen for Aire cofactors reveals a role for Hnrnpl in polymerase release and Aire-activated ectopic transcription.
abstract: Aire induces the expression of a large set of autoantigen genes in the thymus, driving immunological tolerance in maturing T cells. To determine the full spectrum of molecular mechanisms underlying the Aire transactivation function, we screened an AIRE-dependent gene-expression system with a genome-scale lentiviral shRNA library, targeting factors associated with chromatin architecture/function, transcription, and mRNA processing. Fifty-one functional allies were identified, with a preponderance of factors that impact transcriptional elongation compared with initiation, in particular members of the positive transcription elongation factor b (P-TEFb) involved in the release of "paused" RNA polymerases (CCNT2 and HEXIM1); mRNA processing and polyadenylation factors were also highlighted (HNRNPL/F, SFRS1, SFRS3, and CLP1). Aire's functional allies were validated on transfected and endogenous target genes, including the generation of lentigenic knockdown (KD) mice. We uncovered the effect of the splicing factor Hnrnpl on Aire-induced transcription. Transcripts sensitive to the P-TEFb inhibitor flavopiridol were reduced by Hnrnpl knockdown in thymic epithelial cells, independently of their dependence on Aire, therefore indicating a general effect of Hnrnpl on RNA elongation. This conclusion was substantiated by demonstration of HNRNPL interactions with P-TEFb components (CDK9, CCNT2, HEXIM1, and the small 7SK RNA). Aire-containing complexes include 7SK RNA, the latter interaction disrupted by HNRNPL knockdown, suggesting that HNRNPL may partake in delivering inactive P-TEFb to Aire. Thus, these results indicate that mRNA processing factors cooperate with Aire to release stalled polymerases and to activate ectopic expression of autoantigen genes in the thymus.
authors.affiliation: Division of Immunology, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115.
authors.forename: Matthieu
authors.initials: M
authors.lastname: Giraud
authors.forename: Nada
authors.initials: N
authors.lastname: Jmari
authors.forename: Lina
authors.initials: L
authors.lastname: Du
authors.forename: Floriane
authors.initials: F
authors.lastname: Carallis
authors.forename: Thomas J F
authors.initials: TJ
authors.lastname: Nieland
authors.forename: Flor M
authors.initials: FM
authors.lastname: Perez-Campo
authors.forename: Olivier
authors.initials: O
authors.lastname: Bensaude
authors.forename: David E
authors.initials: DE
authors.lastname: Root
authors.forename: Nir
authors.initials: N
authors.lastname: Hacohen
authors.forename: Diane
authors.initials: D
authors.lastname: Mathis
authors.forename: Christophe
authors.initials: C
authors.lastname: Benoist
mesh_terms: D000818:Animals; D002460:Cell Line; D055785:Gene Knockdown Techniques; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000235:genetics / Q000502:physiology*; D006801:Humans; D051379:Mice; D034622:RNA Interference; D014157:Transcription Factors / Q000235:genetics* / Q000502:physiology; D014158:Transcription, Genetic / Q000502:physiology*; D000096927:AIRE Protein
chemical_list: D034441:Heterogeneous-Nuclear Ribonucleoproteins; D014157:Transcription Factors; D000096927:AIRE Protein; C516741:heterogeneous-nuclear ribonucleoprotein I, mouse
keywords: autoimmunity; hnRNP; negative selection