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TAF15 Is Important for Cellular Proliferation and Regulates the Expression of a Subset of Cell Cycle Genes Through Mirnas

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TAF15 Is Important for Cellular Proliferation and Regulates the Expression of a Subset of Cell Cycle Genes Through Mirnas Oncogene (2013) 32, 4646–4655 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc ORIGINAL ARTICLE TAF15 is important for cellular proliferation and regulates the expression of a subset of cell cycle genes through miRNAs M Ballarino1, L Jobert1,4, D Dembe´le´ 1, P de la Grange2, D Auboeuf3 and L Tora1 TAF15 (formerly TAFII68) is a member of the FET (FUS, EWS, TAF15) family of RNA- and DNA-binding proteins whose genes are frequently translocated in sarcomas. By performing global gene expression profiling, we found that TAF15 knockdown affects the expression of a large subset of genes, of which a significant percentage is involved in cell cycle and cell death. In agreement, TAF15 depletion had a growth-inhibitory effect and resulted in increased apoptosis. Among the TAF15-regulated genes, targets of microRNAs (miRNAs) generated from the onco-miR-17 locus were overrepresented, with CDKN1A/p21 being the top miRNAs- targeted gene. Interestingly, the levels of onco-miR-17 locus coded miRNAs (miR-17-5p and miR-20a) were decreased upon TAF15 depletion and shown to affect the post-transcriptional regulation of TAF15-dependent genes, such as CDKN1A/p21. Thus, our results demonstrate that TAF15 is required to regulate gene expression of cell cycle regulatory genes post-transcriptionally through a pathway involving miRNAs. The findings that high TAF15 levels are needed for rapid cellular proliferation and that endogenous TAF15 levels decrease during differentiation strongly suggest that TAF15 is a key regulator of maintaining a highly proliferative rate of cellular homeostasis. Oncogene (2013) 32, 4646–4655; doi:10.1038/onc.2012.490; published online 5 November 2012 Keywords: FUS/EWS/TAF15 (FET) proteins; miRNAs; transcription; proliferation; neuronal differentiation; neuroblastoma INTRODUCTION possible role of TAF15 in additional steps of RNA metabolism. TAF15 (formerly TAFII68) is a nuclear protein known to associate Moreover, an interesting link between FET proteins and miRNAs with a distinct subpopulation of transcription factor IID (TFIID), a has emerged. miRNAs are 21- to 24-nucleotide-long RNA guides multi-subunit complex that nucleates the pre-initiation complex on that regulate the expression of target mRNAs containing the promoters of on protein-coding genes.1,2 As TAF15 was not complementary sequences.9 In animals, miRNA genes are found to be associated with all human TFIID complexes and has no typically transcribed into primary transcripts (pri-miRNA) that ortholog in other non-vertebrate species, TAF15 is not considered a undergo processing by the Drosha-containing complex canonical TAF.3 TAF15 harbors a transcriptional activation domain, Microprocessor. Proteomic studies uncovered that TAF15, a RNA recognition motif and many Arg-Gly-Gly (RGG) repeats together with FUS and EWS, is a subunit of the Microprocessor known to participate in RNA binding.1 TAF15 together with the complex.10 Moreover, the regulation of a group of miRNAs by the related FUS and EWS constitutes the FET (FUS/EWS/TAF15) protein aberrant EWS/Fli protein uncovered a novel mechanism family whose genes are frequently translocated in sarcomas and controlling Ewing’s sarcoma malignancy via miRNAs.11,12 rare hematopoietic and epithelial cancers.4 In each case, the At present, the physiological functions of the wild-type N-terminus of the proteins, containing a potent transcriptional FET proteins, especially that of TAF15, are not well understood, activation domain,5 is fused to the DNA-binding domain of a and no cellular genes regulated by TAF15 have been identified. In transcription factor to form oncogenic chimeras.6 this study, we show that TAF15 depletion has a growth-inhibitory FET proteins have been involved in both transcriptional and effect and increases apoptosis of HeLa cells. By combining post-transcriptional regulatory processes of gene expression, such TAF15-dependent transcriptome analysis with in silico-based as transcription initiation, splicing, DNA repair and RNA matura- miRNA studies, we identified a subset of miRNA targets among tion.6,7 However, to date the majority of studies have focused on TAF15-regulated genes. Indeed, we found that TAF15 is required the functions of FUS and EWS and their aberrant products, but the for the expression of some of the members of the onco-miR-17 role of TAF15 in various cellular metabolisms remains unclear. family and that its depletion leads to miRNA-mediated upregula- Recently, we showed that a chromatin-associated fraction of tion of a target reporter in vivo. Thus, we describe a TAF15- TAF15 associates with a human U1 small nuclear RNA.8 Thus, we dependent gene regulatory network and show that TAF15 is hypothesized that the U1-TAF15 particle is produced by regulating a subset of the cellular transcriptome and protein levels remodeling of the canonical U1-snRNP, further suggesting a via miRNAs. 1Department of Functional Genomics and Cancer, Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, CNRS UMR 7104, INSERM U964, Universite´ de Strasbourg, Illkirch Cedex, France; 2GenoSplice technology, Hoˆpital Saint-Louis, Paris, France and 3INSERM U1052, Cancer Research Center of Lyon, Centre Le´on Be´rard, Lyon, France. Correspondence: Dr L Tora, Department of Functional Genomics and Cancer, Institut de Ge´ne´tique et de Biologie Mole´culaire et Cellulaire, CNRS UMR 7104, INSERM U964, Universite´ de Strasbourg, BP 10142, Illkirch Cedex, 67404 CU de Strasbourg, France. E-mail: [email protected] 4Current address: Biotechnology Center of Oslo, Gaustadallen 21, 0349 Oslo, Norway. Received 21 May 2012; revised 31 August 2012; accepted 7 September 2012; published online 5 November 2012 TAF15 regulates cell cycle and proliferation M Ballarino et al 4647 RESULTS Genome-wide analysis of TAF15-regulated genes TAF15-regulated genes si-LUCsi-TAF15 To gain insight into the function of TAF15 in human cells, we FC >1.5 p-value< 0.05 performed gene expression profiling upon TAF15 knockdown by TAF15 UP DOWN total number RNA interference (Figure 1a). As a control for off-target effects, we regulated regulated GAPDH first generated a stable HeLa GL3 cell line expressing the luciferase 1628 721 907 gene (Supplementary Figure 1a). These cells were transfected in triplicates with small interfering RNAs (siRNAs) directed either against the luciferase mRNA (si-LUC), to eliminate off-target effects 30 mediated by siRNA transfection, or against TAF15 (si-TAF15) si-LUC mRNA. TAF15 silencing was specific, as the si-TAF15 had no effect 25 si-TAF15 B on luciferase activity and efficient at 90% (Supplementary 20 Figures 1a and b and Figure 1a). Total RNA purified from si-TAF15 or si-LUC cells was then used to probe an oligonucleotide-based 15 microarray coding for 26 880 genes. We found that 1628 (7.15%) 10 genes were significantly differently expressed in the two mRNA levels conditions (Figure 1b, Supplementary Table 1): 721 genes were 5 upregulated and 907 were downregulated in si-TAF15 versus 0 si-LUC samples. Real-time quantitative PCR analysis confirmed the FGG TPP1 upregulation (BTG2, FGG) or downregulation (TAF15, TTP1, POLR3B) TAF15 BTG2 CDK6 of genes selected among the 10 most affected genes in each POLR3B GAPDH category (Figure 1c). In the downregulated category, we have also validated CDK6 because of its involvement in cell cycle regulation. Molecular and Cellular Functions 8 A Gene Ontology (GO) study performed with the Ingenuity (IPA) tool assigned the TAF15-regulated genes to a wide set of cellular functions spanning from small molecule biochemistry to protein 6 synthesis and nucleic acid metabolism. However, those genes involved in cell cycle (n ¼ 140; P-value: 2.09 Â 10 À 8–4.63 Â 10 À 3) 4 and cell death (n ¼ 276; P-value: 3.79 Â 10 À 7–4.63 Â 10 À 3) regu- lation were the most highly represented group identified -log(p-value) 2 (Figure 1d) together with the cell growth and proliferation category, which was also statistically significant (P-value: 0 6.9 Â 10 À 5–4.63 Â 10 À 3; Figure 1d). The GO analysis revealed an enrichment of genes regulating the G1/S checkpoint (P-value: 4.14 Â 10 À 3; Supplementary Figures 2a and b) when the Cell Cycle distribution within GO categories was compared with the Cell Death distribution of the whole set of genes represented on the array Cell Signaling (Supplementary Figure 2a). Thus, human TAF15 can both Cell Morphology Drug Metabolism Lipid Metabolism Gene Expression Protein Synthesis Protein Trafficking positively and negatively regulate gene expression, and its Cellular movement Molecular Transport Antigen Presentation Cellular Compromise depletion influences a high proportion of genes involved in cell Cellular Development Amino Acid Metabolism Nucleic Acid Metabolism cycle transition and cell death. Carbohydrate Metabolism Small Molecul Biochemistry Post-translational Modification TAF15 depletion impairs cell proliferation and cell cycle Cellular Growth and Proliferation Cellular Function and Manteinance We next analyzed cell cycle progression and apoptosis in TAF15- Cellular Assembly and Organisation depleted cells. Transfection of HeLa cells with siRNA targeting the Cell-To-Cell Signaling and Interaction coding region of TAF15 mRNA (si-TAF15) resulted in a strong DNA Replication, Recombination, Repair decrease of the protein (Figure 2a, lane 10) 72 h post transfection. Figure 1. TAF15 regulates the expression of specific genes. To investigate whether TAF15 affects viability and cell cycle (a) Western blot analysis shows the levels of TAF15 upon transfection of HeLa GL3 cells with siRNAs directed either against kinetics in vivo, we counted the number of cells at different time TAF15 (si-TAF15) or luciferase (si-LUC) mRNAs. Glyceraldehyde-3- points after siRNAs treatment (Figure 2b). At 72 h post transfec- phosphate dehydrogenase (GAPDH) is used as a loading control. tion, si-TAF15-treated cells showed a 1.4-fold decrease in their (b) Table showing the total number of genes deregulated by TAF15 proliferation rate when compared with control si-LUC cells.
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