Joint Project Leaders:
Günther Schütz, Prof. Dr.
Dep. of Molec. Biol. of the Cell I
German Cancer Res. Center (DKFZ)
Im Neuenheimer Feld 280
D-69120 Heidelberg
Tel. +49. (0) 6221-42 34 11
Alfred Nordheim, Prof. Dr.
Inst. for Cell Biol., Dep. of Molec. Biol.
University of Tübingen
Auf der Morgenstelle 15
D72076 Tübingen
Tel. +49 (0) 7071-297 8898
Fax +49. (0) 6221.42 34 70
E-mail: g.schuetz@dkfz.de
Fax +49 (0) 7071-29 53 59
E-mail: alfred.nordheim@uni-tuebingen.de
Topic: Analyzing and modeling Glucocorticoid Receptor (GR) and Serum
Response Factor (SRF) in hepatocyte-specific gene regulation and signal cross-talk.
Summary: We identified by molecular genetic means the transcriptions factors GR and SRF as two new essential regulatory proteins of hepatocyte function. Combined,
GR and SRF represent transcriptional control modules that direct mRNA expression profiles in response to at least four signaling pathways, namely signaling by steroids,
Stat5-, MAPK, and RhoA/actin. As already initiated with some of the genetically deficient hepatocytes, genome-wide mRNA expression profiling begins to uncover previously unrecognized cross-talk between GR and Stat5 signaling. Altered mRNA expression profiles in GR-, Stat-5, and SRF-deficient adult hepatocytes will be investigated in normal liver physiology and during liver regeneration. Obtained gene profiles and signal cascade activation states will be modeled over time and differing physiological states (e. g. liver regeneration) to obtain new systems biological insight into hepatocyte function.
Introduction:
Glucocorticoid receptor (GR): Our major focus is to analyze how steroid hormones and their receptors control gene expression in both liver development and adult hepatocyte physiology. GR regulates transcription through DNA binding and crosstalk with other transcription factors, e.g. Stat5. We found that GR activity in hepatocytes is essential for normal postnatal growth of mice (Tronche et al. 2004).
Immunoprecipitation (IP) and chromatin-IP of GR suggested that the effect of the receptor is due to its interaction with Stat5, which is a critical mediator of growth hormone (GH) signaling. This reveals an important role of Stat5-GR interactions in vivo , specifically in controlling postnatal body growth. Interestingly, hepatocytespecific Stat5 null mice display a similar growth deficiency as GR null mice. To investigate the importance of GR/Stat5 interactions in hepatocyte-specific transcription, we compared the mRNA expression profiles in wildtype versus GR- or
Stat5-deficient hepatocytes. Interestingly, we uncovered that a large proportion
(about 1/3) of the GR and Stat5 target genes are shared (unpublished), suggesting that coactivation of Stat5 by GR is a common mode of both glucocorticoid action and
GH-dependent growth regulation.
Serum response factor (SRF): SRF-dependent target genes are subject to regulation by two types of intracellular signaling cascade, namely signaling by MAPK and by Rho-dependent actin dynamics. Our previous work suggests SRF to have an essential role in hepatocytes during embryonal liver development. This observation fits our prior understanding of SRF controlling migration and survival of embryonic cells. It remains to be investigated at what stage in embryonic liver development and for what molecular (or cellular) reason hepatocyte-specific SRF-deficiency leads to embryonic lethality. Also, the role of SRF in adult hepatocyte physiology needs to be

investigated, specifically with regard to the process of liver regeneration. This study will reveal the relevance of SRF-directed MAPK signaling and RhoA/actin signaling, both during liver development and adult liver growth and regeneration.
Workplan:
All GR/Stat5- and SRF-associated data sets to be obtained (mRNA profiles, activity of signaling cascades), will be suitable for systems biological evaluation and modeling (see below).
GR: To determine the general impact of GR/Stat5 interaction on the entire liver transcriptome, including the expression of microRNAs, we will extend our initial
Affymetrix microarray studies (covering 12.500 sequences) to analyse the complete transcriptome at different developmental and adult phases of wt versus GR- and
Stat5-deficient livers. Dynamic changes in clusters of GR/Stat5 co-regulated genes will be identified during peri- and post-natal, as well as adult stages of liver growth.
We will focus on the role of the interaction of the GR/Stat5 transcription factors in glucose, fatty acid, and lipid metabolism, with particular emphasis on the development of steatosis and detoxification reactions. This will be analyzed by comparing gene expression profiles in wild type versus GR mutants defective in dimerization (i.e. mutants unable to bind DNA but still able to cooperate with Stat5) in comparison to the hepatocyte-specific GR null mutant (i.e. defective in both DNA binding and Stat5 cooperation). Furthermore, consecutive stages during liver regeneration upon partial hepatectomy will be investigated in collaboration with R.
Gebhardt, Leipzig. The role of the transcription factors SRF, CREB, GR and Stat5 during liver regeneration following partial hepatectomy will be analyzed exploiting hepatocyte-specific mutants generated with the Cre/loxP system in the respective genes. Since mice with hepatocyte-restricted loss of SRF die during fetal development, we will use a fusion protein of the Cre recombinase (Cre-ERT2) with a modified ligand binding domain of the estradiol receptor to generate time-controlled, ligand inducible mutations. This methodology has been successfully exploited in our laboratories to generate inducible mutations in e.g. dopaminergic and noradrenergic neurons. We are currently generating mice expressing Cre-ERT2 in a liver specific fashion. Since we anticipate the transcription factors under investigation to be active in the early phase of liver regeneration, we will focus on the analysis of target genes by expression profiling in the first four hours after hepatectomy. To dissect the complex transcriptional control, computational methods for elucidation of transcription factor binding sites will be exploited (in collaboration with H.P. Herzel, Berlin and J.
Timmer, Freiburg). Further, we will not restrict our expression analysis to traditional protein coding sequences but also assess profiles of more than 100 microRNAs using qPCR and investigate roles of microRNAs in liver development and physiology.
SRF: The precise stage of embryonic lethality, as associated with developing SRFdeficient livers, will be determined and the associated liver mRNA expression (versus embryonic wt liver) will be profiled (Affymetrix). To circumvent embryonic lethality, an inducible, hepatocyte-specific Cre-expressing mouse will be generated (Alfp-
CreERT2, see above) to induce SRF-deficiency in adult livers and to establish differential (wt vs. SRF-deficient) adult hepatocyte expression profiles. Furthermore, consecutive stages during liver regeneration (subsequent to partial hepatectomy) will be investigated (collaborator: Prof. Gebhardt, Leipzig). All expression profiling data will be supplemented with kinetic activity profiles of SRF-directed signaling cascades
(MAPK, RhoA/actin), using sets of activity-specific antisera.
Modelling: Genome wide differential target gene expression profiles (Affymetrix
GeneChips) and kinetic changes in acitivity states of signaling cascades that involve

the transcription factors GR, Stat5, CREB, and SRF will be modeled (J. Timmer,
Freiburg; Herzel, Berlin).
Milestones per Year:
GR:
2007: - entire hepatic transcriptome during postnatal development: wt vs. liver- specific Stat5 and GR mutation
- analysis of microRNAs
2008: - hepatic transcriptome in adults: wt vs. liver-specific Stat5 and GR mutation analysis of hepatic transcriptome in GRdim and GRnls mutants
2009: - transcriptome analysis after hepatectomy in wt and mutants
SRF:
2007: - generating Alfp-CreERT2 mice
- determine liver defects of SRF-deficient embryos
- determine liver-specific phenotypes of adult SRF deficiency in liver
2008: - diff. mRNA & cascade act. profiles (wt vs. SRF-def. embr./adult livers)
2009: - diff. mRNA & cascade act. profiles (wt vs. SRF-def. regener. livers)
Modelling:
2007-2009 – Modeling of mRNA expression profiles and signaling cascades targeting the transcription factors GR, Stat5, CREB, and SRF during hepatectomy.
Budget:
GR: personal: 1x BATVb,
consumables: € 15.000,- / p.a.
SRF: personal: 1x BATIIa/2, consumables: € 20.000,-/p.a.
350 mice: € 9.100,- / p.a. 350 mice: € 9.100,- / p.a.
These costs are resulting from the extensive use of Affymetrix (GeneChip) and
Applied Biosystems (qRT-PCR) technologies. Also, costly antibodies for quantitation of signaling activities need to be purchased. Additionally, mouse husbandry requires a significant budget (€ -,50 / mouse / week).
References:
• Tronche, F.; Opherk, C.; Moriggl, R.; Kellendonk, C.; Reimann, A.; Schwake, L.; Reichardt,
H. M.; Stangl, K.; Gau, D.; Hoeflich, A.; Beug, H.; Schmid, W.; Schütz, G. (2004).
Glucocorticoid receptor function in hepatocytes is essential to promote postnatal body growth. Genes Dev 18 , 492-497.
• Knöll, B., Kretz, O., Fiedler, C., Alberti, S., Schütz, G., Frotscher, M. and Nordheim, A.
(2006). Serum Response Factor controls neuronal circuit assembly in the hippocampus. Nat
Neurosci. 9 , 195-204.
• Opherk, C.,Tronche, F., Kellendonk, C., Kohlmüller, D., Schulze, A., Schmid, W., and
Schütz, G. (2004) Inactivation of the glucocorticoid receptor in hepatocytes leads to fasting hypoglycemia and ameliorates hyperglycemia in streptozotocin-induced diabetes mellitus.
Mol. Endocrinol. 18, 1346-1353 .
• Kenzelmann, M., Hergenhahn, M., Maertens, S., K ueffer, S., Klären, R., Hotz-Wagenblatt,
A., Ittrich, C., Li, L., Wang, S., Volz, M., Lemberger, T., Arribas-Prat, R., Jonnakuty, S.,
Hollstein, M., Schmid, W., Gretz, N., Gröne, H.-J., and Schütz, G. (2005). Non-invasive invitro and in-vivo analysis of de-novo transcribed RNA. Nat. Methods (in revision).
• Alberti, S., Krause, S.M., Kretz, O., Philippar, U., Lemberger, T., Casanova, E., Wiebel,
F.F., Schwarz, H., Frotscher, M., Schütz, G., and Nordheim, A., (2005). Neuronal migration in the murine rostral migratory stream requires serum response factor, PNAS 102, 6148-
6153.