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Division of Molecular Biology

Department of Medical Biochemistry

Division of Molecular Biology

Research Groups

 

Transcriptional Regulation by Members of the E2F-
and SP-Families of Transcription Factors

Group Leader:         Hans Rotheneder

Postdocs:

PhD Students:          Barbara Galos

Diploma students:    Ludwig Schwarzmayr
                                Peter Andorfer

Technician:             

   |    Lectures   |

Cooperative Activation of Growth Induced Promoters

E2F is a family of heterodimeric transcription factors which regulate genes, whose products are essential for progression through the mammalian cell cycle. One of those is the thymidine kinase gene. The promoter of the murine thymidine kinase gene carries one binding site for E2F and one for the well known transcription factor Sp1. In vivo binding of both sites is abolished by mutation of either one of the binding sites. The binding sites are separated by ten basepairs only which suggested an interaction between the two factors. We have recently shown that a subgroup of the E2F family members are able to interact directly with Sp1 (Karlseder et al., 1996). The domain which mediates the binding of Sp1 is present only in E2F1, -2 and -3 but not in E2F4 and -5. Closer examination of this region revealed that amino acids 102-122 of E2F1 are necessary and sufficient for the binding of Sp1. This is close to and slightly overlapping with the binding site for cyclin A which has been mapped to amino acids 67-108. In vitro binding of the two proteins to E2F1 is independent and non-interfering with each other. Within Sp1 the zinc-finger region which is located in the carboxy-terminal part is required for the interaction with E2F. Recently it was reported that Sp1 also belongs to a family of closely related factors. Among these transcription factors Sp2 shows the least homology of all family members and has a different DNA binding specificity. The expression of Sp4 is restricted to the brain and neural tissue. Sp3, ubiquitously expressed like Sp1 has been described as a negatively acting factor competing with Sp1 for DNA binding sites. In Sp1 knock out mice on the other hand it seems to take over most of the functions of Sp1. We studied the properties of Sp2, Sp3 and Sp4 and found that all four Sp proteins are able to bind E2F1. Sp1, Sp3 and, to a lesser extent Sp4, but not Sp2 are able to activate the murine thymidine kinase promoter in mammalian as well as in insect cells (Rotheneder et al,1999). Synergistic activation with E2F1 however occurs only in mammalian cells, suggesting a requirement for species-specific cofactors and/or chromatin structure.

Regulation of Sp1 Activity

The study of transcriptional regulatory mechanisms has demonstrated the critical role for promoter-binding proteins that are essential for full activity of a given gene. Many of these transcription factors are composed of independent domains which define DNA-binding specificity, activate transcription or mediate interaction with other proteins. Sp1, a ubiquitously expressed protein of 95-105 kDa that binds DNA through C-terminal zinc finger motifs, stimulates transcription from promoters containing a G+C rich sequence, the GC box.
Sp1 exhibits multiple domains which seem to be important for different aspects of its activity. The N-terminal half of Sp1 harbours two glutamine rich regions (domains A and B) which mediate transcriptional activation. Domain C maps to a segment of high charge density just N-terminal relative to the zinc fingers. Domain D, located at the extreme C-terminal end of the protein mediates interaction with other Sp1 molecules and is thereby necessary for the synergistic activation of promoters containing more than one binding site. Whereas the protein level is rather constant in most tissues, the activity of Sp1 seems to be tightly controlled by posttranslational modifications and interactions with regulatory proteins.
We have found that histone deacetylase 1 (HDAC-1) a negative regulator of growth is able to interact with Sp1 thereby changing it into a repressor of transcription. HDAC-1 binds to the zinc finger region of Sp1 like E2F-1. Co-expressed E2F-1 physically displaces HDAC-1 from Sp1 thus relieving HDAC mediated repression of Sp1 activity. (Doetzlhofer et al, 1999). This suggests that that the zinc finger region of Sp1 is not only neccessary for DNA binding but also for the control of Sp1 activity by mediating the binding of regulatory proteins.

Isolation of Proteins Interacting with the Amino-terminal
Domains of E2F-1, E2F-2, and E2F-3

Many biologically important processes involve protein-protein interactions. To study such processes proteins can be used as targets for the isolation of cDNA's encoding their prospective partners. Today the most widely used assay is the yeast two hybrid system also called "interaction trap. This method utilizes hybrid genes to detect protein-protein interactions via the activation of reporter-gene expression. The amino terminal regions of E2F-1, -2, and -3 seem to be indispensable for the downregulation of these factors during the S-phase. Cells expressing E2F-1devoid of the amino terminal 87 amino acids exhibit an extremely rounded morphology and lack detectable microfilaments and microtubules. Furthermore E2F-1 has been shown to activate the promoter of the herpesvirus simplex thymidine kinase (HSV-TK) in the absence of a consensus E2F binding site. Contrary to the activation through consensus E2F sites the upregulation of the HSV-TK promoter required the entire E2F-1 including the sequences N-terminal of the DNA-binding domain. Sequences close to the DNA binding domain of E2F-1, -2, and -3 mediate binding to Sp1. Experiments in our laboratory have shown that the amino terminal domain of E2F-1 is able to bind several proteins out of an extract from in vivo labeled cells. This suggests that the amino terminal domains of E2F-1, -2, and -3 play a significant albeit largely undefined role in the regulation of this subgroup of the E2F family. Protein-protein interactions with yet unknown factors migth determine promoter specificity and/or activity in development, cell cycle and differentiation. These proteins can be transcription factors like Sp1 or regulatory factors like cyclinA/cdk2. Identifying, cloning and characterising these proteins will lead to a better understanding of the machinery controlling crucial processes of the cell via the E2F family of transcription factors.

Publications
  1. Knofler M, Saleh L, Bauer S, Galos B, Rotheneder H, Husslein P, Helmer H.(2004). Transcriptional regulation of the human chorionic gonadotropin beta gene during villous trophoblast differentiation. Endocrinology. 2004 Apr;145(4):1685-94. Epub 2004 Jan 08.

  2. Lagger,G., Doetzlhofer,A., Schuettengruber,B., Haidweger,E., Simboeck,E., Tischler,J., Chiocca,S., Suske,G., Rotheneder,H., Wintersberger,E., and Seiser,C. (2003)
    The tumor suppressor p53 and the deacetylase HDAC1 are antagonistic regulators of the Cyclin-dependent Kinase inhibitor p21/WAF1/CIP1 gene. Mol. Cell. Biol. Vol.23, No.8, 2669-2679

  3. Vasicek R, Meinhardt G, Haidweger E, Rotheneder H, Husslein P, Knofler M. (2003). Expression of the human Hand1 gene in trophoblastic cells is transcriptionally regulated by activating and repressing specificity protein (Sp)-elements. Gene 2003 Jan 2; 302(1-2):115-27.

  4. Haidweger, E., Novy, M., and Rotheneder, H. (2001). Modulation of Sp1 Activity by a Cyclin A/CDK Complex. J.Mol. Biol. (306), 201-212.

  5. Rotheneder, H., Geymayer, S. & Haidweger, E. (1999). Transcription factors of the Sp1 family: Interaction with E2F and regulation of the murine thymidine kinase promoter. J.Mol. Biol. (293), 1005-1015.

  6. Doetzlhofer, A., Rotheneder, H., Lagger, G., Koranda, M., Kurtev, V., Brosch, G., Wintersberger, E., and Seiser, C. (1999). Histone deacetylase 1 can repress transcription by binding to Sp1. Mol Cell Biol 19 19 (8), 5504-5511.

  7. Karlseder, J., Rotheneder, H., and Wintersberger, E. (1996). Interaction of Sp1 with the growth- and cell cycle-regulated transcription factor E2F. Mol Cell. Biol. 16, 1659-1667.

Diploma Theses:
Sibylle Geymayer (1997), Eva Haidweger (1998), Tina Weiland (2001), Regina Pohn (2002), Ferner-Ortner Judith (2004)

PhD Theses:
Eva Haidweger (2001), Michael Novy (2002)

Collaborations:
Erhard Wintersberger, Christian Seiser (this department) and Martin Knöfler, Universitätsklinik für Frauenheilkunde (AKH).

   

"EAPP": A novel E2F1 associated nuclear phosphoprotein. (U2OS cells)

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