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Different models have been proposed explaining how eukaryotic gene transcription is terminated. Recently, Nsi1, a factor involved in silencing of ribosomal DNA (rDNA), was shown to be required for efficient termination of rDNA transcription by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae. Nsi1 contains Myb-like DNA binding domains and associates in vivo near the 3= end of rRNA genes to rDNA, but information about which and how DNA sequences might influence Nsi1-dependent termination is lacking. Here, we show that binding of Nsi1 to a stretch of 11 nucleotides in the correct orientation was sufficient to pause elongating Pol I shortly upstream of the Nsi1 binding site and to release the transcripts in vitro. The same minimal DNA element triggered Nsi1-dependent termination of pre-rRNA synthesis using an in vivo reporter assay. Termination efficiency in the in vivo system could be enhanced by inclusion of specific DNA sequences downstream of the Nsi1 binding site. These data and the finding that Nsi1 blocks efficiently only Pol I-dependent RNA synthesis in an in vitro transcription system improve our understanding of a unique mechanism of transcription termination. C ellular multisubunit RNA polymerases of all three domains of life share common structural and functional features. After initiation of transcription at their cognate gene promoters, they extend the transcripts until they reach specific DNA cis elements, at which transcription is terminated. Termination occurs when the contacts of the RNA-DNA hybrid within the elongating RNA polymerase are destabilized. Termination finally results in the stopping of RNA synthesis, release of the transcript, and dissociation of the RNA polymerase from the DNA template (reviewed in reference 1). A first step in the termination pathway can be the pausing of the elongating polymerase, which is then followed by the disruption of the elongation complex.Dissociation of the ternary elongation complex formed by RNA polymerase, RNA, and DNA can be caused either by auxiliary protein factors or solely by interactions of DNA and RNA with the transcribing RNA polymerase. Both principles, factordependent and intrinsic termination, have been described for bacterial transcription termination (reviewed in reference 1). In the case of eukaryotic RNA polymerase II (Pol II), either 3=-end processing factors of the mRNA or the yeast RNA-DNA helicase Sen1 (human senataxin) was suggested to disrupt the ternary complex (reviewed in references 2 and 3). This might resemble Rho-dependent termination in bacteria in which the ATP-dependent RNA-DNA helicase Rho destabilizes the elongation complex (4, 5; for a review, see reference 1). RNA polymerase III (Pol III) termination resembles the other bacteria-like termination mechanism, the intrinsic termination, in which the RNA-DNA hybrid is destabilized by a stem-loop in the nascent RNA (6, 7) (8).In contrast to these mechanisms of transcription termination, Pol I requires a termination factor which binds to a specific DNA sequence t...

About one third of the known cellular oncogenes are transcription factors (TFs). As many tumors are dependent on continuous activity of these proteins they represent highly interesting targets for therapeutic approaches e.g. by inhibition with small molecule inhibitors. Because of lack of enzymatic activity and accompanied difficulty of direct targeting, TF have been considered undruggable for a long time. This view recently changed with the demonstration that, instead of direct targeting, indirect targeting of protein partners required for TF activity might represent an appropriate alternative. Among transcription factors targeted by this approach are Notch, Bcl6 and EWS/FLI, the oncogenic fusion protein in Ewing sarcoma. In our studies we focus on alveolar Rhabdomyosarcoma (aRMS), an aggressive and highly metastatic pediatric sarcoma. ARMS is characterised by the expression of the fusion TF PAX3/FOXO1 which is essential for the survival of aRMS cells. Here, we aimed to identify interacting proteins that are involved in modulating the aberrant activity of PAX3/FOXO1 in aRMS. Towards this end, we purified the interactome of PAX3/FOXO1 from two different aRMS cell lines and identified the individual interactors by mass spectrometry. This led to the description of 150 candidate interactors which were reproducibly co-purified with PAX3/FOXO1 in four independent experiments. Most of the identified proteins are transcription factors or transcriptional regulators. To validate our findings, selected candidate interactors such as RUNX1 and TFAP2B were further shown to pull down PAX3/FOXO1 in co-immunoprecipitation studies. To analyse whether some of the candidate interactors have an influence on PAX3/FOXO1 activity we individually silenced the expression of a set of 60 candidates by siRNA and measured the expression of several PAX3/FOXO1 target genes including TFAP2B, FGFR4, and NMYC. These experiments revealed that interactors can have different effects on specific PAX3/FOXO1 target genes and suggest that specific transcriptional complexes are responsible for activating the transcription of different PAX3/FOXO1 target genes. This potentially reflects the diversity of mechanisms by which PAX3/FOXO1 regulates target gene expression including enhancer- and promoter-directed actions. Further characterization of selected interactors is necessary to understand the impact of these on the oncogenic function of PAX3/FOXO1 in more detail. The results of these experiments shed light on the regulatory pathway(s) and mechanisms involved in PAX3/FOXO1 mediated oncogenesis and thereby contribute to our understanding of the oncogenic function of tumor-specific chimaeric transcription factors. Citation Format: Maria Böhm, Marco Wachtel, Beat W. Schäfer. Identification of the interactome of PAX3/FOXO1 in alveolar rhabdomyosarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 757. doi:10.1158/1538-7445.AM2013-757

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