title: Sequence-function correlations and dynamics of ERG isoforms. ERG8 is the black sheep of the family.
abstract: The transcription factor ERG is known to have divergent roles. On one hand, it acts as differentiation factor of endothelial cells. On the other hand, it has pathological roles in various cancers. Genomic analyses of the ERG gene show that it gives rise to several isoforms. However, functional differences between these isoforms, representing potential reasons for distinct effects in diverse cell types have not been addressed in detail so far. We set out to investigate the major protein isoforms and found that ERG8 contains a unique C-terminus. This isoform, when expressed as GFP-fusion protein, localized mainly to the cytosol, whereas the other major isoforms (ERG1-4) were predominantly nuclear. Using site directed mutagenesis and laser scanning microscopy of live cells, we could identify nuclear localization (NLS) and nuclear export sequences (NES). These analyses indicated that ERG8 lacks a classical NLS and the DNA-binding domain, but holds an additional NES within its distinctive C-terminus. All the tested isoforms were shuttling between nucleus and cytosol and showed a high degree of mobility. ERG's 1 to 4 were transcriptionally active on ERG-promoter elements whereas ERG8 was inactive, which is in line with the absence of a DNA-binding domain. Fluorescence resonance energy transfer (FRET) microscopy revealed that ERG8 can bind to the transcriptionally active ERG's. Knockdown of ERG8 in endothelial cells resulted in upregulation of endogenous ERG-transcriptional activity implying ERG8 as an inhibitor of the active ERG isoforms. Quantitative PCR revealed a different ratio of active ERG's to ERG8 in cancer- versus non-transformed cells.
authors.affiliation: Dept. of Vascular Biology and Thrombosis Research, Center for Physiology and Pharmacology, Medical University of Vienna, Austria.
authors.forename: Bastian
authors.initials: B
authors.lastname: Hoesel
authors.affiliation: Dept. of Vascular Biology and Thrombosis Research, Center for Physiology and Pharmacology, Medical University of Vienna, Austria.
authors.forename: Naila
authors.initials: N
authors.lastname: Malkani
authors.affiliation: Dept. of Vascular Biology and Thrombosis Research, Center for Physiology and Pharmacology, Medical University of Vienna, Austria.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Hochreiter
authors.affiliation: Dept. of Vascular Biology and Thrombosis Research, Center for Physiology and Pharmacology, Medical University of Vienna, Austria.
authors.forename: José
authors.initials: J
authors.lastname: Basílio
authors.affiliation: Dept. of Vascular Biology and Thrombosis Research, Center for Physiology and Pharmacology, Medical University of Vienna, Austria.
authors.forename: Kalsoom
authors.initials: K
authors.lastname: Sughra
authors.affiliation: Dept. of Vascular Biology and Thrombosis Research, Center for Physiology and Pharmacology, Medical University of Vienna, Austria.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Ilyas
authors.affiliation: Dept. of Vascular Biology and Thrombosis Research, Center for Physiology and Pharmacology, Medical University of Vienna, Austria.
authors.forename: Johannes A
authors.initials: JA
authors.lastname: Schmid
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D049790:Nuclear Export Signals / Q000235:genetics*; D019913:Nuclear Localization Signals / Q000235:genetics*; D011485:Protein Binding; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000071230:Transcriptional Regulator ERG
chemical_list: C072312:ERG protein, human; D049790:Nuclear Export Signals; D019913:Nuclear Localization Signals; D020033:Protein Isoforms; D015534:Trans-Activators; D000071230:Transcriptional Regulator ERG; D049452:Green Fluorescent Proteins
keywords: DNA binding protein; ERG; ETS transcription factor family; Fluorescence recovery after photobleaching (FRAP); Fluorescence resonance energy transfer (FRET); Microscopy; Nuclear export sequence; Nuclear localization sequence