Arno Helmberg

Publications (5)24.06 Total impact

• Article: A RAS recruitment screen identifies ZKSCAN4 as a glucocorticoid receptor-interacting protein.

  Karin Ecker, Andreas Lorenz, Frank Wolf, Christian Ploner, Günther Böck, Tod Duncan, Stephan Geley, Arno Helmberg
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  ABSTRACT: To search for proteins interacting with the glucocorticoid receptor, we adapted Aronheim's reverse RAS recruitment system relying on the Saccharomyces cerevisiae mutant cdc25-2, which has a temperature-dependent defect in its RAS signaling pathway driving proliferation. The full-length human glucocorticoid receptor (NR3C1, isoform-alpha) was attached to the yeast plasma membrane in either of two orientations and used as bait to screen a HeLa cell cDNA library. Library proteins were fused to constitutively active, soluble human RAS, complementing the defective yeast pathway in case of bait-prey interaction. Screening of 800 000 clones resulted in the isolation of 21 proteins, 8 of which were followed up to evaluate interaction with the receptor in human cell lines. One of these candidates, the SCAN- and KRAB-domain-containing zinc finger protein 307 (ZKSCAN4) was co-precipitated with the receptor when both proteins were overexpressed in HEK293 cells. Rabbit antisera against ZKSCAN4 were raised, affinity purified, and used to immunoprecipitate endogenous ZKSCAN4 from Hct116 cells, resulting in co-precipitation of endogenous glucocorticoid receptor. Overexpressed ZKSCAN4 was found to co-localize in granular nuclear structures with the activated glucocorticoid receptor and partially with chromatin regions characterized by histone H3 mono-methylated on lysine 4 (H3K4me1). Overexpressed ZKSCAN4 had no effect on an episomal glucocorticoid receptor-driven reporter plasmid. By contrast, ZKSCAN4 markedly reduced glucocorticoid induction of the mouse mammary tumor virus-promoter-driven reporter gene when this was chromosomally integrated, arguing for a chromatin-dependent inhibition of glucocorticoid receptor-mediated transactivation.
  Journal of Molecular Endocrinology 12/2008; 42(2):105-17. · 3.48 Impact Factor
• Article: Identification, tissue expression, and glucocorticoid responsiveness of alternative first exons of the human glucocorticoid receptor.

  Elisabeth Presul, Stefan Schmidt, Reinhard Kofler, Arno Helmberg
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  ABSTRACT: Transcripts for the human glucocorticoid receptor (NR3C1) are known to contain alternative first exons 1A1, 1A2, and 1A3 from the distal promoter or 1D, 1E, 1B, 1F, 1C, or 1H from the proximal promoter. Here, we report two additional alternative first exons identified by Rapid amplification of cDNA ends (RACE)-PCR. The first, exon 1I, starts approximately 700 bp downstream of the splice donor site of the longest form of exon 1A, 1A3, considerably extending the known distal promoter region with a region containing conserved transcription factor-binding sites as well as a potential glucocorticoid response element (GRE) that differs from the consensus GRE in only two positions. The second, exon 1J, is part of the proximal promoter region and resides between exons 1D and 1E. Since this has been determined by quantitative real-time reverse transcriptase (RT)-PCR, exon 1I is used foremost in cells of the T-lymphocyte lineage. In the T-ALL cell line CEM-C7H2, which is sensitive to glucocorticoid-induced apoptosis, transcripts containing alternative first exons from the distal as well as the proximal promoter regions were markedly autoinduced by glucocorticoid treatment, with more pronounced relative induction in the distal promoter. Neither transcript was autoinduced in the related, resistant cell lines CEM-C1, and CEM-C7R5. In contrast, the glucocorticoid-sensitive PreB697 cell line strongly autoinduced transcripts from the proximal promoter, but not transcripts from the distal promoter, to relevant levels. Therefore, the autoinductive feedback loop implicated in glucocorticoid-induced apoptosis cannot universally rely on the distal promoter of the glucocorticoid receptor.
  Journal of Molecular Endocrinology 03/2007; 38(1-2):79-90. · 3.48 Impact Factor
• Article: Mouse Variable‐Region Gene Families: Complexity, Polymorphism and Use in non‐Autoimmune Responses

  Reinhard Kofler, Stephan Geley, Heinz Kofler, Arno Helmberg
  Immunological Reviews 04/2006; 128(1):5 - 21. · 11.15 Impact Factor
• Article: Gene expression profiling of human endothelial cells exposed to 50-Hz magnetic fields fails to produce regulated candidate genes.

  Blair Henderson, Michaela Kind, Guenther Boeck, Arno Helmberg, Georg Wick
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  ABSTRACT: To address the question of a possible effect of magnetic fields (MF) at 50 Hz on living systems, gene expression analyses were performed on human primary vascular endothelial cells exposed to MF of various intensities compared to control cells. Exposure protocols included continuous exposure at a single intensity (10 and 700 microT), intermittent exposure at a single intensity (700 microT), and continuous exposure to a variable-intensity field (10-30 microT). The transcriptional response of the cells was investigated using oligonucleotide microarrays containing up to 30 000 unique features. Although in individual experiments genes were identified where the expression appeared to be affected by exposure to MF, none of these genes were regulated in the same manner in subsequent repetition experiments. This is the first report of a transcriptome-wide analysis of the effects of MF exposure on human cells. The lack of a reproducible effect of MF on the expression of any genes in our investigation adds further weight to the evidence that 50-Hz MF are not capable of interacting with biological systems and thus do not represent an endothelial stress factor.
  Cell Stress and Chaperones 02/2006; 11(3):227-32. · 3.01 Impact Factor
• Article: Mouse V k gene classification by nucleic acid sequence similarity

  Robert Strohal, Arno Helmberg, Guido Kroemer, Reinhard Kofler
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  ABSTRACT: Analyses of immunoglobulin (Ig) variable (V) region gene usage in the immune response, estimates ofV gene germline complexity, and other nucleic acid hybridization-based studies depend on the extent to which such genes are related (i. e., sequence similarity) and their organization in gene families. While mouseIgh heavy chainV region (V H ) gene families are relatively well-established, a corresponding systematic classification ofIgk light chainV region (V k ) genes has not been reported. The present analysis, in the course of which we reviewed the known extent of theV k germline gene repertoire andV k gene usage in a variety of responses to foreign and self antigens, provides a classification of mouseV k genes in gene families composed of members with >80% overall nucleic acid sequence similarity. This classification differed in several aspects from that ofV H genes: only someV k gene families were as clearly separated (by >25% sequence dissimilarity) as typicalV H gene families; mostV k gene families were closely related and, in several instances, members from different families were very similar (>80%) over large sequence portions; frequently, classification by nucleic acid sequence similarity diverged from existing classifications based on amino-terminal protein sequence similarity. Our data have implications forV k gene analyses by nucleic acid hybridization and describe potentially important differences in sequence organization betweenV H andV k genes.
  Immunogenetics 11/1989; 30(6):475-493. · 2.93 Impact Factor