An RNAi screen for Aire cofactors reveals a role for Hnrnpl in polymerase release and Aire-activated ectopic transcription Matthieu Girauda,b, Nada Jmarib, Lina Dua, Floriane Carallisb, Thomas J. F. Nielandc, Flor M. Perez-Campod, Olivier Bensaudee, David E. Rootc, Nir Hacohenc, Diane Mathisa,1, and Christophe Benoista,1 aDivision of Immunology, Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA 02115; bDepartment of Immunology, Institut Cochin, Institut National de la Santé et de la Recherche Médicale (INSERM) U1016, Université Paris Descartes, 75014 Paris, France; cThe Broad Institute of MIT and Harvard, Cambridge, MA 02142; dDepartment of Internal Medicine, Hospital U.M. Valdecilla-Instituto de Formación e Investigación Marqués de Valdecilla, University of Cantabria, 39008 Santander, Spain; and eEcole Normale Supérieure, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8197, INSERM U1024, 75005 Paris, France Contributed by Christophe Benoist, December 19, 2013 (sent for review December 3, 2013) Aire induces the expression of a large set of autoantigen genes in Aire primarily impacts the elongation steps of transcription, in the thymus, driving immunological tolerance in maturing T cells. particular by releasing promoter-bound polymerases that remain To determine the full spectrum of molecular mechanisms underlying paused after abortive initiation (8, 9). Correspondingly, Aire has the Aire transactivation function, we screened an AIRE-dependent been shown to interact with subunits of the key controller of gene-expression system with a genome-scale lentiviral shRNA polymerase release, the positive transcription elongation factor library, targeting factors associated with chromatin architecture/ b (P-TEFb) (8, 10, 11). P-TEFb is recruited to stalled initiation function, transcription, and mRNA processing. Fifty-one functional complexes in many systems of regulated transcription. It phos- allies were identified, with a preponderance of factors that impact phorylates Ser2 on the C-terminal domain (CTD) of Pol-II and transcriptional elongation compared with initiation, in particular the elongation inhibitors DRB sensitivity-inducing factor (DSIF) members of the positive transcription elongation factor b (P-TEFb) and negative elongation factor (NELF), dislodging NELF and involved in the release of “paused” RNA polymerases (CCNT2 and converting DSIF into an activator (12–14). P-TEFb itself exists in IMMUNOLOGY HEXIM1); mRNA processing and polyadenylation factors were also an inactive form when bound to the 7SK small nuclear RNA or highlighted (HNRNPL/F, SFRS1, SFRS3, and CLP1). Aire’s functional to HEXIM1 (15, 16). allies were validated on transfected and endogenous target genes, To better understand the molecular mechanisms that Aire including the generation of lentigenic knockdown (KD) mice. We exploits to promote ectopic gene expression, and to complement uncovered the effect of the splicing factor Hnrnpl on Aire-induced the biochemical interaction analyses (7), we undertook a sys- transcription. Transcripts sensitive to the P-TEFb inhibitor flavopiridol tematic large-scale RNAi screen to identify Aire’s transcriptional were reduced by Hnrnpl knockdown in thymic epithelial cells, in- allies. The results confirmed the importance of factors involved dependently of their dependence on Aire, therefore indicating in transcriptional elongation and in RNA processing and iden- a general effect of Hnrnpl on RNA elongation. This conclusion tified several previously unrecognized elements of the Aire was substantiated by demonstration of HNRNPL interactions pathway, which we validated in RNAi knockdown (KD) lenti- with P-TEFb components (CDK9, CCNT2, HEXIM1, and the small genic mice. We also identified physical interaction and func- 7SK RNA). Aire-containing complexes include 7SK RNA, the latter tional involvement of RNA splicing regulators, in particular interaction disrupted by HNRNPL knockdown, suggesting that Hnrnpl, in elongation control, helping to integrate the diverse HNRNPL may partake in delivering inactive P-TEFb to Aire. Thus, classes of Aire-associated proteins previously uncovered. these results indicate that mRNA processing factors cooperate with Aire to release stalled polymerases and to activate ectopic Results expression of autoantigen genes in the thymus. Identification of AIRE’s Functional Allies by a High-Throughput RNAi Lentivirus Screen. To perform the intended RNAi screen for Aire autoimmunity | negative selection | hnRNP allies, we needed to develop a robust assay for Aire-induced gene expression, with a strong signal of Aire activity, minimal noise to he transcription factor Aire plays a very specific role in the Tmanagement of immunological tolerance, promoting the ec- Significance topic expression, in medullary epithelial cells (MECs) of the thymus, of a wide array of peripheral-tissue antigens (PTA) (1). The transcription factor Aire controls an unusual mode of Differentiating T cells are thus exposed to the self-antigens that transcriptional regulation, important to establish immune tol- they will encounter later on, and potentially self-reactive cells are erance to self, which allows the ectopic expression in the thy- deleted or deviated to regulatory phenotypes as a result. Aire- mic epithelium of RNA transcripts normally restricted to defined deficient mice and human patients develop autoantibodies and tissues. Through a genome-scale RNAi, we identify 51 func- multiorgan autoimmune infiltration, similarly to human patients tional partners of Aire, reaffirming a role for Aire in unleashing with a mutation at the AIRE gene locus (reviewed in ref. 2). stalled transcription, interestingly through involvement of RNA Aire is an unusual transcriptional regulator. It impacts thou- processing factors. sands of PTA-encoding genes whose expression is very differ- ently controlled in parenchymal tissues (3), does not have a clear Author contributions: M.G., N.J., T.J.F.N., F.M.P.-C., O.B., D.E.R., N.H., D.M., and C.B. de- signed research; M.G., N.J., L.D., F.C., T.J.F.N., and F.M.P.-C. performed research; M.G., D.M., DNA binding motif, adjusts its targets according to the cell in and C.B. analyzed data; and M.G., D.M., and C.B. wrote the paper. which it is expressed (4), and takes as cues nonspecific marks of The authors declare no conflict of interest. inactive chromatin such as unmethylated H3K4 (5, 6). Indeed, 1To whom correspondence may be addressed. E-mail: [email protected] or dm@hms. Aire-interacting proteins include factors involved in chromatin harvard.edu. structure/modification, transcriptional elongation, and pre-mRNA This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. processing (7). Several lines of investigation have shown that 1073/pnas.1323535111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1323535111 PNAS Early Edition | 1of6 Downloaded by guest on September 30, 2021 avoid masking true signals, and amenable to high throughput. It Firefly Luciferase AIRE A Luminescence (RLU) B was impossible to use primary MECs, given their very low yield AIRE 0 2 4 6 8 - from a mouse thymus, so we opted for transient transfection of luc pGL3 CHRNA1 + AIRE-sensitive AIRE-resistant an AIRE-expression construct into the AIRE-negative human promoter - luc CHRNA1-pGL3 minP CMV MEC line 4D6 (17). An assay of AIRE-induced transcription + Firefly Renilla - Renilla luc2 pGL4 based on a paired chemiluminescence readout (firefly/ + luciferase) was implemented. Expanding from our earlier expe- TATAbox - luc2 minP-pGL4+ Transfection rience (18), we tested a panel of luciferase reporter plasmids RNAi AIRE ’ lentivirus library Luminescence cotransfected with into 4D6 cells and found that AIRE s Renilla Luciferase detection Luminescence (RLU) Renilla Infection Firefly / impact was highest for plasmids with largely inactive minimal 0 4 8 12 16 promoters (human CHRNA1, or an isolated TATA box; Fig. SV40 T7 - SV40-phRL 72h 16h6h 1A). In contrast, AIRE did not increase luciferase expression hRluc + CMV T7 - 4D6 cells puromycin luc driven by strong eukaryotic promoters (SV40 or CMV). hR CMV-phRL+ MEC selection To validate the specificity of the assay, we verified that transactivation of minP-luciferase luminescence by AIRE did C Transcriptional regulators + RNA binding factors correspond to increased reporter mRNA (Fig. S1A), and that 15065 shRNAs, 2994 genes Controls Aire expression constructs bearing deletions of certain of the P = NS Aire functional domains were indeed impaired in luciferase in- 4 duction (Fig. S1B). The screen strategy is outlined in Fig. 1B: infection of 4D6 cells by a lentivirus library in the LKO.1 vector 2 encoding short hairpin RNAs (shRNAs) under the U6 promoter (one virus per well in 384-well plates); selection of transduced 0 Z score cells by puromycin treatment; cotransfection with the AIRE- Z0.05 -2 expressing construct, an AIRE-dependent (minimal promoter -1.5 driven) firefly luciferase reporter and an AIRE-insensitive (CMV -4 promoter driven) Renilla luciferase reporter; and quantitation of 227 genes P < 0.007 Renilla luminescence from the two reporters. luciferase provided 0 5000 10000 15000 0 340 an internal control for shRNAs that would generically affect shRNA number transcription or cell viability and served as a normalization for AIRE secondary screen D E Z0.05 transfection efficiency, which varied with the density of puro- 227 genes -1.4 mycin-surviving cells and thus with the titer of each lentivirus