The estrogen receptor (ER) is a ligand-activated transcription factor that regulates gene expression. The classical mechanisms of nuclear ER action include ligand-induced dimerization of ER which binds estrogen responsive elements (EREs) in promoters of target genes. In addition, non-genomic pathways of ER action have also been identified in breast cancer cells. Cdc25A is a tyrosine phosphatase that catalyzes dephosphorylation of cyclin/cyclin-dependent kinase complexes to regulate G1- to S-phase cell cycle progression. Cdc25A mRNA levels are induced by 17β-estradiol (E2) in ZR-75 breast cancer cells, and deletion analysis of the Cdc25A promoter identified the -151 to -12 region as the minimal E2-responsive sequence. Subsequent mutation/deletion analysis showed that at least three different cis-elements were involved in activation of Cdc25A by E2, namely, GC-rich Sp1 binding sites, CCAAT motifs, and E2F sites. Studies with inhibitors and dominant negative expression plasmids show that E2 activates Cdc25A expression through activation of genomic ERα/Sp1 and E2F1 and cAMP-dependent activation of NF-YA. Thus, both genomic and non-genomic pathways of estrogen action are involved in induction of Cdc25A in breast cancer cells. The PIAS family was initially identified as cytokine-induced inhibitors of STATs which contain several conserved domains involved in binding to other nuclear coactivators. In this study we have investigated coactivation of ERα by PIAS3 in breast cancer cell lines transiently cotransfected with the pERE3 constructs which contain three tandem EREs linked to a luciferase reporter gene. PIAS3 coactivated ERα-mediated transactivation in cells cotransfected with pERE3 and wild-type ERα. In contrast to many other coactivators, PIAS3 also enhanced transactivation of ERα when cells were cotransfected with the TAF1 ERα mutant. In addition, PIAS3 does not interact with activation function 2 (AF2) domain of ERα in a mammalian two-hybrid assay. These data indicate that coactivation of ERα by PIAS3 was AF2-domain independent. Analysis of several PIAS3 deletion mutants showed that the region containing amino acids 274 to 416 of PIAS3 are required for coactivation suggesting that the RING finger domain and acidic region of PIAS3 are important for interactions with wild-type ERα. These results demonstrate that PIAS3 coactivated ERα and this represents a non-classical LXXLL-independent coactivation pathway.

Der Transkriptionsfaktor CCAAT/Enhancer Binding Protein beta (C/EBPbeta) reguliert die Genexpression, Proliferation und Differenzierung in verschiedenen Zelltypen. Die Funktion von C/EBPbeta wird durch Interaktionen mit einer Reihe von Kofaktoren moduliert, die Bestandteile von Chromatin-verŠndernden oder Transkriptions-regulierenden makromolekularen Maschinen sind. Die Identifikation und funktionelle Charakterisierung dieser Kofaktoren trŠgt ma§geblich zum VerstŠndnis der Biologie von C/EBPbeta bei. C/EBPbeta wird zudem in vielfŠltiger Weise posttranslational reguliert. Beispielsweise kann C/EBPbeta phosphoryliert, SUMOyliert, acetyliert und an mehreren Positionen an Arginin- und Lysinresten methyliert werden. Die SUMOylierung von C/EBPbeta gilt als SchlŸsselmodifikation, die nachfolgende Modifikationen steuert und zu einer VerŠnderung der genregulatorischen Eigenschaften von C/EBPbeta fŸhrt. C/EBPbeta bindet an zwei Enzyme der SUMOylierungsmaschinerie, Ubc9 und PIAS3. Es konnte gezeigt werden, dass PIAS3 nicht nur als E3-Ligase bei der SUMOylierungsreaktion dient, sondern auch mit SUMO-modifiziertem C/EBPbeta interagieren und als transkriptioneller Repressor wirken kann. Um weitere Interaktionspartner von C/EBPbeta zu identifizieren wurde ein System zur Proteom-weiten Identifikation von Bindungspartnern etabliert. Dazu wurden radioaktiv markierbare Proteinsonden hergestellt, welche die Identifikation von Bindungspartnern auf Protein-Macroarrays ermšglichten. Neben der transaktivierenden DomŠne (TAD) wurde die regulatorische Region in ihrer SUMOylierten und nicht-modifizierten Form in Screening-Experimenten eingesetzt. Eine Vielzahl von neuen C/EBPbeta-Bindungspartnern konnte identifiziert werden, wobei die konstitutive SUMOylierung C/EBPbeta-Interaktionen verŠndern kann. Bei den identifizierten Proteinen handelt es sich um Mitglieder der Polycomb Gruppe, Chromatin-modifizierende Enzyme, SignaltransduktionsmolekŸle und transkriptionelle Koregulatoren. Wissenschaftlich besonders interessant war die Identifikation der Lysin-Methyltransferase H3-K9-HMTase 3 (G9a) als Bindungspartner der transaktivierenden Region von C/EBPbeta. Diese Interaktion wurde durch GST-Bindungs- und KoimmunoprŠzipitationsstudien bestŠtigt. Durch massenspektrometrische Analysen konnte Monomethylierung der AminosŠuren K39 und K168 in C/EBPbeta nachgewiesen werden. Dadurch ergab sich die Vermutung, dass G9a nicht nur die Methylierung von Histon H3 katalysiert, sondern auch fŸr die Methylierung und Regulation von C/EBPbeta verantwortlich ist. Rekombinantes C/EBPbeta konnte durch G9a in vitro methyliert werden. Koexpression von C/EBPbeta und G9a fŸhrte zu einer Reduktion der transaktivierenden Eigenschaften von C/EBPbeta in AbhŠngigkeit von der katalytischen SET-DomŠne von G9a. Dieser Reduktion konnte durch Mutation der AminosŠuren K39 und K168 in Alanin entgegengewirkt werden. Als Bindungspartner der C/EBPbeta TAD konnte au§erdem die intrazellulŠre DomŠne von Notch1 (Notch1-ICD) identifiziert werden. Der Notch-Signalweg ist ein evolutionŠr konservierter Genschalter, der an vielen Entscheidungen in der Entwicklung sowie bei physiologischen und pathophysiologischen Prozessen im adulten Organismus, wie z. B. akuter lymphatischer T-Zell LeukŠmie (T-ALL), beteiligt ist. Die Interaktion zwischen Notch1-ICD und C/EBPbeta konnte in GST-Bindungsexperimenten und KoimmunoprŠzipitationsstudien verifiziert werden. In Reportergenstudien wurde eine Stimulation der C/EBPbeta-abhŠngigen Transkription durch Notch1-ICD beobachtet. C/EBPbeta ist demnach ein ZielmolekŸl des Notch1-Signalweges. / The transcription factor CCAAT/Enhancer Binding Protein beta (C/EBPbeta) regulates gene expression, proliferation and differentiation of various cell types. The function of C/EBPbeta is modulated by a number of co-factors which are components of macromolecular machines that alter the state of chromatin or that regulate gene transcription. Identification and functional characterisation of these co-factors is crucial for understanding the biology of C/EBPbeta. C/EBPbeta is regulated by a number of posttranslational modifications and can be found in phosphorylated, SUMOylated, acetylated and arginine- or lysine-methylated forms. SUMOylation of C/EBPbeta is considered a key modification which controls subsequent modifications. These modifications alter the gene regulatory functions of C/EBPbeta. C/EBPbeta binds two enzymes of the SUMOylation machinery, Ubc9 and PIAS3. This study shows that PIAS3 not only has E3-ligase activity during the SUMOylation of C/EBPbeta, but also interacts with SUMO-modified C/EBPbeta leading to repression of transcription. A proteome-wide screening procedure was established to identify novel interaction partners of C/EBPbeta. It was based on radioactively labelled proteins that can be utilized as probes to identify binding partners on solid phase protein-macroarrays. The C/EBPbeta transactivation domain (TAD) and its regulatory region in SUMOylated and non-SUMOylated form were used in different screening approaches. Using this procedure a number of novel C/EBPbeta interaction partners were identified, that depended in part on the SUMOylation status of C/EBPbeta. The major part of the C/EBPbeta-interacting proteins are members of the Polycomb group, chromatin-modifying enzymes, signal transduction molecules and transcriptional co-regulators. Interestingly, the lysine-methyltransferase H3-K9-HMTase 3 (G9a) was among the binding partners of C/EBPbeta that interacted with the TAD. This interaction was verified by GST-pulldown and co-immunoprecipitation studies. Mass spectrometrical analysis identified the amino acids K39 and K168 of C/EBPbeta to be mono-methylated. Therefore it was speculated that G9a not only catalyzes the methylation of Histone H3 but may also methylate and regulate C/EBPbeta. Indeed, recombinant C/EBPbeta could be methylated by G9a in vitro. Co-expression of C/EBPbeta and G9a resulted in a reduction of the transactivating potential of C/EBPbeta, which depended on the catalytical SET domain of G9a. This reduction could be counteracted by mutating the amino acids K39 and K168 to alanine. In addition to G9a, the Notch1 intracellular domain (Notch1-ICD) could also be identified as a novel binding partner of the C/EBPbeta TAD. Notch is a component of an evolutionary conserved pathway that acts on numerous physiological and pathophysiological processes during development and in the adult, e.g. T-cell acute lymphoblastic leukemia (T-ALL). The interaction between Notch1-ICD and C/EBPbeta could be verified in GST-pulldown studies and by co-immunoprecipitation. Reporter gene studies showed a stimulation of C/EBPbeta-dependent transcription through Notch1-ICD. C/EBPbeta is therefore a novel target molecule of the Notch1 signaling pathway.