Background Transcription elements of the CSL (CBF1/RBP-Jk/Suppressor of Hairless/LAG-1) family are key regulators of metazoan development and function as the effector components of the Notch receptor signalling pathway implicated in various cell fate She decisions. conserved arginine residue abolishes DNA binding in both CSL paralogs similar to the situation in mouse. We have also demonstrated the ability of Cbf11 and Cbf12 to activate gene expression in an autologous fission yeast reporter system. Conclusions/Significance Our results indicate that the fission yeast CSL proteins are indeed genuine family members capable of functioning as transcription factors and provide support for the ancient evolutionary origin of this important protein family. Introduction Transcription factors of the CSL (CBF1/RBP-Jk/Suppressor of Hairless/LAG-1) family belong among key regulators of metazoan development. They are context-dependent activators or repressors of gene expression and function as the effector components of the Notch receptor signalling pathway BTZ038 required for various cell differentiation-related decisions [1]-[3]. Defects in Notch/CSL signalling have been implicated in numerous human diseases including several types of cancer [4] [5]. Apart from their role in Notch signalling Notch-independent BTZ038 functions in gene regulation have also been described for CSL proteins and RBP-L one of the two mammalian CSL paralogs appears to operate completely independently of Notch [6]-[8]. CSL target genes typically contain a well-defined sequence motif (GTG[G/A]AA) in their regulatory regions which is bound specifically by CSL proteins [9]-[11]. Several CSL mutants compromised in their ability to bind DNA have been reported [12] and the crystal structure of the CSL bound to DNA has provided a rationale to explain the effects of these mutations and to describe the mode of DNA binding in CSL family members [13]. In BTZ038 our previous studies we have identified a number of novel putative members of the CSL protein family in various fungal species [14] [15] i.e. in organisms lacking the other Notch pathway components [16]. We have shown that Cbf11 and Cbf12 the CSL paralogs in the fission yeast and open reading frame and 20 nt complementary to the ends of the tagging cassette. The PCR product was gel-purified transformed into cells and nourseothricin-resistant clones in which the cassette had been integrated by homologous recombination were selected as described [22]. Table 2 Oligonucleotides used in this study. A list of plasmids used in this study can be found in Table 3. All plasmids for CSL overexpression were based on the pREP41/42 vector series for N-terminal EGFP HA or MycHis tagging which contain the medium-strength thiamine-regulated promoter version [23]. The Cbf11(Δ1-172) Cbf12(Δ1-465) and Cbf12(395-465) truncations were cloned by PCR using the High Fidelity PCR Enzyme Mix or Taq (Fermentas) TA or TOPO TA BTZ038 Cloning Kit (Invitrogen) suitable primers and fission yeast genomic DNA or previously constructed plasmids containing full-length CSL cDNAs as templates [17]. CSL variants with a DNA binding mutation (DBM) in the beta-trefoil domain were constructed using the QuickChange II site-directed mutagenesis kit (Agilent) and the indicated primers. All new CSL variants were verified by sequencing. Table 3 Plasmids used in this study. β-galactosidase reporter plasmids were derived from pREP42EGFPN. The part of the promoter upstream BTZ038 of the TATA box which is responsible for the thiamine-dependent regulation of expression was removed (up to the PstI site). The attenuated TATA box (“gene was PCR-cloned from the drosophila pCasper-AUG-betaGal vector and fused in frame (SalI/BamHI) to GFP contained in the modified pREP42EGFPN vector. Finally double stranded DNA oligonucleotides (derived from EMSA probes) with NheI-compatible overhangs were inserted into the NheI site and their number and orientation were determined by a combination of restriction digestion analysis PCR and sequencing. Microscopy Live cells overexpressing EGFP fusions of CSL protein variants were immobilized on a glass slide by a thin layer of agarose gel and subjected to fluorescence microscopy using an Olympus CellR system. Images were analysed with imageJ. Protein sequence analysis Protein sequence conservation was assessed using ClustalW [24]. Nuclear localization signals (NLS) were searched for using PredictProtein [25] NLStradamus [26] and cNLS Mapper [27] with BTZ038 defaults settings. Western blotting Proteins were separated on a 7.5% Tris-glycine polyacrylamide gel transferred to a nitrocellulose membrane and probed with either an alkaline phosphatase-conjugated goat polyclonal anti-GFP (ab6661 Abcam; 1∶1200.