Department of Medical Biochemistry, Division of Molecular Biology


Dominique Meunier	Gordin Zupkovitz	Reinhard Grausenburger	Reinhard Brunmeir	Elisabeth Simböck


Stefan Winter	Jana Pulkertova	Sabine Lagger	Jenny Jurkin	Martina Schuster


Mascha Rauscher

We are part of the GEN-AU project.

EPIGENETIC PLASTICITY OF THE MAMMALIAN GENOME

Lagger et al. (2002) EMBO J. 21:2672-2681

Fig 1. HDAC1 is essential for mouse embryonic devlopment. HDAC1 wildtype and null embryo at E9.5. HDAC1 is highly expressed throughout the E9.5 embryo except the developing heart (he) with pronounced expression in the bronchial arches (ba) and the limb bud (lb).
RESEARCH INTERESTS
Background

The DNA of eukaryotic cells is organized as nuclear chromatin. The basic unit of this structure, the nucleosome consists of 146 bp DNA wrapped around a histone octamer. The highly conserved amino-terminal tails of core histones are the targets of multiple post-translational modifications including methylation, phosphorylation and acetylation of which the latter is certainly the best studied. Acetylation of core histones is believed to correlate with transcriptionally active chromatin. Dynamic histone acetylation/deacetylation was shown to be important for different cellular processes like replication, gene silencing, position effect variegation, X chromosome inactivation and differentiation. We are in particular interested in the characterization of mammalian histone modifying enzymes and their functions in gene expression, proliferation and development.

Current projects
Chromatin Structure and Gene Expression: The Role of Histone deacetylase 1

We have identified murine histone deacetylase 1 (HDAC1) as a growth-factor regulated enzyme (Bartl et al. (1997)). Interestingly, HDAC1 protein levels vary up to ten-fold in different mouse cell lines indicating the existence of a cell type-specific balance between acetylating and deacetylating activities. Overexpression of HDAC1 in mouse fibroblasts leads to a severe delay during the G2/M phases of the cell cycle and dramatic changes in nuclear morphology (Bartl et al. (1997)). Similarly, loss of HDAC1 also results in a cell cycle phenotype (Lagger et al., (2002)) suggesting that HDAC1 expression and activity has to be tightly controlled.Transcription of the HDAC1 gene is activated by growth factors and this activation is mediated by events involving the cooperative acetylation and phosphorylation of core histones (Hauser et al., (2002)). This mechanism allows the induction of the HDAC1 gene by growth stimuli and the feedback regulation dependent on the intracellular levels of HDAC1 (Schuettengruber, et al., (2003)).
Previously, it was shown that repression by transcriptional inhibitors like Mad/Max, members of the retinoblastoma protein family and several nuclear receptors is in part mediated by the targeting of histone deacetylases to specific promoter regions. Aberrant recruitment of histone deacetylases by PML-RAR, PLZF-RAR and AML1-ETO was observed in leukemia caused by chromosomal translocations. In this context we want to examine the molecular and biological role of HDAC1 during growth activation, cell cycle and differentiation of mammalian cells. We have started to identify HDAC1-interacting proteins, which recruit the enzyme to specific target promoters or modulate its activity. In this way we have identified the transcription factor Sp1 as HDAC1-associated protein (Doetzlhofer et al., (1999); Lagger et al., (2003)). Sp1 seems act as an anchor for both positive and negative regulators of transcription and is therefore required for both activation and repression of particular target genes.
Recently, in collaboration with other research groups we have identified Evi-1 (Vinatzer et al., (2001)) and the viral protein Gam-1 (Chiocca et al., (2002)) as HDAC1-binding factors. While Evi-1 acts as a transcriptional repressor by recruiting HDAC1 to target promoters, the adenovirus product Gam-1 inactivates the deacetylase to facilitate virus replication by inducing specific host genes. Gam-1 also interferes with sumoylation of HDAC1 (Colombo et al., (2002)). We have generated a panel of acetyl specific antibodies directed against each of the four target lysine residues of histone H4 (Taplick et al., 1998). Using this set of specific antisera and commercially available antibodies we investigate by chromatin immunoprecipitation the acetylation state of nucleosomes within specific target promoters. In this way we would like to establish a direct link between the recruitment of HDAC1 by specific transcription factors to certain target promoters and local changes in the chromatin structure of these promoters.

Role of Histone deacetylase 1 for proliferation and development.

In a complementary approach we are currently studying the role of HDAC1 during embryonic development. HDAC1 is expressed early during mouse embryogenesis (Figs. 1 and 2) and might therefore be important for developmental processes. Indeed, disruption of the HDAC1 gene results in embryonic lethality due to developmental defects and impaired proliferation (Lagger et al., (2002)). One of the HDAC1 target genes is the CDK inhibitor p21/WAF1, that is up-regulated in HDAC1 null cells (Lagger et al., (2002)). This prompted us to analyze the role of HDAC1 in the regulation of the p21 gene in human tumor cells. This study showed thatthe tumor suppressor p53 and the deacetylase HDAC1 are antagonistic regulators of p21 gene expression (Lagger et al., (2003)). Taken together we propose that HDAC1 is required for mouse embryonic development and unrestricted proliferation of mammalian cells.

Fig 2. HDAC1 expression during mouse preimplantation development. Mouse preimplantation embryos from the 1-cell to the blastocyst stage have been immunolabelled for histone deacetylase HDAC1 (red channel) and stained with 4', 6-diamidino-2-phenylindole (DAPI, blue channel). Low levels of HDAC1 can be detected in the pronuclei of the fertilized oocyte. High-level expression starts in the 2-cell stage at the time when zygotic genome activation occurs and stays high throughout further development until birth (data not shown).

REFERENCES 2005

V. Kurtev, R. Margueron, K. Kroboth, E. Ogris, V. Cavailles and C. Seiser (2004).
Transcriptional regulation by the repressor of estrogen receptor activity via recruitment of histone deacetylases. J. Biol. Chem. 279:24834-43

Schuettengruber,B., Simboeck,E., Khier, H. and Seiser,C. (2003)
Autoregulation of Histone deacetylase 1 Expression. Mol. Cell. Biol. 23:6993-7004

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

Schuettengruber,B., Doetzlhofer,A., Kroboth,K., Wintersberger,E., and Seiser,C. (2003)
Alternate activation of two divergently transcribed mouse genes from a bidirectional promoter is linked to changes in histone modification. J. Biol. Chem. 278:1784-93.

Hauser,C., Schuettengruber,B., Bartl,S., Lagger,G. and Seiser,C. (2002)
Activation of the HDAC1 gene by cooperative histone phosphorylation and acetylation. Mol. Cell. Biol. 22:7820-7830

Lagger, G., O’Carroll, D., Rembold, M., Khier, H., Weitzer, G., Tischler, J., Jenuwein, T. and Seiser, C. (2002)
Essential function of histone deacetylase 1 in proliferation control and CDK inhibitor repression. EMBO J. 21:2672-2681.

Taplick J., Kurtev, V., Kroboth, K., Posch, M., Lechner, T. and Seiser,C. (2001)
Homo-oligomerisation and nuclear localisation of mouse HDAC1. J. Mol. Biol. 308:27-38.

Posch, M., Hauser, C., and Seiser C. (2000)
Substrate Binding is a Prerequisite for Stabilisation of Mouse Thymidine Kinase in Proliferating Fibroblasts.
J. Mol. Biol. 300: 493-502

Khier, H., S. Bartl, B. Schuettengruber and C. Seiser (1999)
Cloning and characterization of the mouse histone deacetylase 1 gene: Integration of a retrovirus in 129SV mice. Biochim. Biochim. Biophys. Acta 1489: 365-373

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

Posch, M., H. Sutterluety, T. Skern and C. Seiser (1999)
Characterization of the Translation-dependent Step during Iron- regulated Decay of Transferrin Receptor mRNA J. Biol. Chem. 274: 16611-16618

Taplick, J., V. Kurtev, G. Lagger and C. Seiser (1998)
Histone H4 acetylation during interleukin-2 stimulation of mouse T-cells.
FEBS Letters 436:349-352

Sutterluety, H., S. Bartl, A. Doetzlhofer, H. Khier, E. Wintersberger and C. Seiser (1998)
Growth-regulated antisense transcription of the mouse thymidine kinase gene.
Nucl. Acids Res. 26:4989-4995

Sutterluety, H. and C. Seiser (1997)
Thymidine inhibits the growth arrest-specific degradation of thymidine kinase protein in transfected L fibroblasts.
J. Mol. Biol., 265:153-160

Bartl, S., J. Taplick, G. Lagger, H. Khier, K. Kuchler and C. Seiser (1997)
Identification of mouse histone deacetylase 1 as a growth factor inducible gene.
Mol. Cell. Biol., 17:5033-5043

Collaborations

Vietor,I., Vadivelu,S.K., Wick,N., Hoffman,R., Cotton,M., Seiser,C., Fialka,I., Wunderlich,W., Bister,M., Haase,A., Korinkova,G., Brosch,G., and Huber,L.A. (2002)
TIS7 is a novel component of the mammalian SIN3 histone deacetylase complex.
EMBO J. 21:4621-31
Colombo,R., Boggio,R., Seiser,C., Draetta,G.F. and Chiocca,S. (2002) The adenovirus protein Gam1 interferes with sumoylation of histone deacetylase 1.
EMBO Rep. 3:1062-1068
Chiocca,S., Kurtev,V., Colombo,R., Sciurpi, M., Brosch, G., Seiser,C., Draetta,G. and Cotten, M. (2002).
Histone deacetylase 1 is sequestered and inactivated by an adenovirus early gene product.
Curr. Biol. 12:594-8..
Vinatzer,U., Taplick, J., Seiser,C., Fonatsch, C. and Wieser, R. (2001).
The leukemia-associated transcription factors EVI-1 and MDS1/EVI1 repress transcription by interacting with histone deacetylase.
Br. J. Haematol. 114:566-73.
Kolle, D., G. Brosch, T. Lechner, A. Pipal, W. Heiliger, J. Taplick and P. Loidl (1999)
Different types of maize histone deacetylases are distinguished by a highly complex substrate and site specificity.
Biochemistry 38(21):6769-6773
Bauer,A., W. Mikulits, G. Lagger, G. Stengl, G. Brosch and H. Beug (1998)
The thyroid hormone receptor functions as a ligand-operated developmental switch between proliferation and differentiation of erythroid progenitors.
EMBO Journal 17:4291-4303.
Grignani,F., S. De Matteis, C. Nervi, L. Tomassoni, V. Gelmetti, M. Cioce, M. Fanelli, M. Ruthardt, F.F. Ferrara, I. Zamir, C. Seiser, F. Grignani, M.A. Lazar, S. Minucci and P.-G. Pelicci (1998)
Fusion proteins of the retinoic acid receptor-alpha recruit histone deacetylase in promyelocytic leukemia.
Nature 391:815-818
Sommer, A., S. Hilfenhaus, A. Menkel, E. Kremmer, C. Seiser, P. Loidl and B. Lüscher (1997)
Cell growth inhibition by the Mad/Max complex through recruitment of histone deacetylase activity.
Curr. Biol. 7:357-365

DIPLOMA AND PH.D. THESES
Diploma Theses: Lukas Orel (1995), Markus Posch (1995), Dagmar Essl (1996), Christoph Hauser (1998), Harald Khier (1999), Gerda Lagger (1998), Vladislav Kurtev (1998), Bernd Schuettengruber (1999), Martina Rembold (2001), Karin Kroboth (2001), Julia Tischler (2002), Elisabeth (Lilli) Simboeck (2003), Reinhard Brunmeir (2003), Stefan Winter (2004), Sarah Keusch (2004)
Ph.D. Theses: Hedwig Sutterluety (1995), Stefan Bartl (1998), Markus Posch (1999), Jan Taplick (1999), Gerda Lagger (2001), Christoph Hauser (2001), Vladislav Kurtev (2002), Bernd Schuettengruber (2002)

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Division of Molecular Biology

Department of Medical Biochemistry

Division of Molecular Biology

Research Groups

CHROMATIN, GENE EXPRESSION AND DEVELOPMENT