Dataset Information

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.

SUBMITTER: Giraud M

PROVIDER: S-EPMC3910647 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

An RNAi screen for Aire cofactors reveals a role for Hnrnpl in polymerase release and Aire-activated ectopic transcription.

Giraud Matthieu M Jmari Nada N Du Lina L Carallis Floriane F Nieland Thomas J F TJ Perez-Campo Flor M FM Bensaude Olivier O Root David E DE Hacohen Nir N Mathis Diane D Benoist Christophe C

Proceedings of the National Academy of Sciences of the United States of America 20140116 4

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 ...[more]

PMID: 24434558

Similar Datasets

Project description:Resistance to tamoxifen in breast cancer patients is a serious therapeutic problem and major efforts are underway to understand underlying mechanisms. Resistance can be either intrinsic or acquired. We derived a series of subcloned MCF7 cell lines that were either highly sensitive or naturally resistant to tamoxifen and studied the factors that lead to drug resistance. Gene-expression studies revealed a signature of 67 genes that differentially respond to tamoxifen in sensitive vs. resistant subclones, which also predicts disease-free survival in tamoxifen-treated patients. High-throughput cell-based screens, in which >500 human kinases were independently ectopically expressed, identified 31 kinases that conferred drug resistance on sensitive cells. One of these, HSPB8, was also in the expression signature and, by itself, predicted poor clinical outcome in one cohort of patients. Further studies revealed that HSPB8 protected MCF7 cells from tamoxifen and blocked autophagy. Moreover, silencing HSBP8 induced autophagy and caused cell death. Tamoxifen itself induced autophagy in sensitive cells but not in resistant ones, and tamoxifen-resistant cells were sensitive to the induction of autophagy by other drugs. These results may point to an important role for autophagy in the sensitivity to tamoxifen. For defined estrogen culture experiments, sensitive (MCF7-B7TamS) and resistant (MCF7-G11TamR) subclones, were plated in 10-cm dish (Nunc) in DMEM supplemented with 5% FBS. The day after plating, the media was changed to steroid depleted media (phenol red-free DMEM) supplemented with 5% charcoal-dextran treated FBS and grown for 2 d. On day 3, cells were rinsed three times with PBS and treated in triplicate using one of four different conditions: (i) estrogen-depleted medium (control); (ii) estrogen (E) at 10 to 9 M; (iii) 4-OHT at 1 uM; and (iv) E plus 4-OHT, both drugs at the same concentrations as in (ii) and (iii). After 4 h, RNA was isolated and processed for gene-expression profiling according to the Affymetrix protocol (Human Genome U133 plus 2.0 Array) at the Microarray Core Facility at the Dana Farber Cancer Institute. The gene-expressionCEL files were normalized using dChip, using the invariant method and PM-MM difference methods for background subtraction. We identified genes that are differentially regulated by tamoxifen using the following criteria: (i) Genes that responded to estrogen in both MCF7-B7TamS andMCF7-G11TamR were identified if cells under tamoxifen treatment were more than 1.1-fold either up- or down-regulated [90% confidence interval (CI)] relative to its corresponding control cells; (ii) Genes unresponsive to tamoxifen in resistant cells: genes that showed more than 1.1-fold up- or down-regulation (90% CI) in MCF7-G11TamR but not in MCF7-B7TamS when comparing cells treated with both estradiol and tamoxifen with those treated with drug only.

Dataset Information

An RNAi screen for Aire cofactors reveals a role for Hnrnpl in polymerase release and Aire-activated ectopic transcription.

Publications

An RNAi screen for Aire cofactors reveals a role for Hnrnpl in polymerase release and Aire-activated ectopic transcription.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets