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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.

The CCAAT binding-factor (CBF) is a transcriptional activator conserved in eukaryotes. The CBF in Saccharomyces cerevisiae is a multimeric heteromer termed the Hap2/3/4/5 complex. Hap4, which contains the activation domain of the complex, is also the regulatory subunit and is known to be transcriptionally controlled by carbon sources. However, little is known about Hap4 regulation. In this report, I identify mechanisms by which Hap4 is regulated, including: (1) transcriptional regulation via two short upstream open reading frames (uORFs) in the 5' leader sequence of HAP4 mRNA; (2) proteasome-dependent degradation of Hap4; and (3) identification of two negative regulators of HAP4 expression, CYC8 and SIN4. I also report differential patterns of Hap4 cellular localization which depends on (1) carbon sources, (2) abundance of Hap4 protein, and (3) presence or absence of mitochondrial DNA (mtDNA).

Activation of tissue-specific genes is a tightly controlled process that normally involves the combined action of several transcription factors and transcriptional co-regulators. The bone-specific osteoca1cin gene (OC) has been used as a prototype to study both tissue-specific and hormonal responsiveness. In this study we have examined the role of Runx2, VDR and C/EBP factors in the regulation of OC gene transcription. Contributions of the Runx and VDRE motifs to OC promoter activity were addressed by introducing point mutations within the context of the rat (-1.1 kb) osteocalcin promoter fused to a CAT-reporter gene. The functional significance of these mutations was assayed following transient transfection and after genomic integration in ROS 17/2.8 osteoblastic cell lines. Furthermore, we tested the effect of these mutations on the chromatin organization of the OC promoter. Our data show that all three Runx sites are required for maximal activation of the OC promoter and that the distal sites contribute significantly to the basal activity. Strikingly, mutation of the three Runx sites abrogates responsiveness of the OC promoter to vitamin D; this loss is also observed when only the Runx sites flanking the VDRE are mutated. Chromatin changes that result in the appearance of DNase I hypersensitive sites during activation of the OC gene are well documented. Mutation of the three Runx sites results in altered chromatin structure as reflected by absence of DNase I hypersensitive sites at the vitamin D response element and over the proximal, tissue-specific basal promoter. These data are consistent with the critical role of Runx2 in osteoblast maturation and bone development. Mutation of the VDRE resulted in a complete loss of vitamin D responsiveness; however, this mutant promoter exhibited increased basal activity. The two DNase I hypersensitive sites characteristic of the transcriptionally active OC gene in osteoblastics cells were not altered upon mutation of the VDRE element, although restriction enzyme accessibility in the proximal promoter region was decreased. We also found an increased level of histone H3 acetylation at the VDRE mutant promoter in comparison to the endogenous gene. Thus binding of VDR to OC promoter is required to achieve a normal transcriptional regulation and chromatin structure of the OC gene. Although Runx2 is considered a master gene for bone development and osteoblast differentiation, it is noteworthy that osteoblast-specific transcription of the rat OC promoter occurs even in the absence of Runx sites. Therefore, other transcription factor(s) should be able to drive OC expression. We characterized a C/EBP enhancer element in the proximal promoter of the rat osteoca1cin gene that resides in close proximity to a Runx element, essential for tissue-specific activation. We find that C/EBPβ or δ and Runx2 factors interact together in a synergistic manner to enhance OC transcription in cell culture systems. Mutational analysis demonstrated that this synergism is mediated through the C/EBP responsive element in the OC promoter and requires a direct interaction between Runx2 and C/EBPβ or δ. Taken together, our findings strongly support a mechanism in which combinatorial interaction of Runx2, VDR, C/EBPβ or δ and probably other transcription factors are needed for regulating OC expression. In this process Runx factors not only act as simple transcriptional trans activators but also by facilitating modifications in promoter architecture and maintaining an active conformation of the target gene promoter.

Chromatin has a compact organization in which most DNA sequences are structurally inaccessible and functionally inactive. Reconfiguration of thechromatir required to activate transcription. This reconfiguration is achieved by the action of enzymes that covalently modify nucleosomal core histones, and by enzymes that disrupt histone-DNA interactions via ATP hydrolysis. TheSWI/SNF family of ATP-dependent chromatin remodeling enzymes has been implicated not only in gene activation but also in numerous cellular processes including differentiation, gene repression, cell cycle control, recombination and DNA repair. PPARγ, C/EBPα and C/EBPβ are transcription factors with well established roles in adipogenesis. Ectopical expression of each of these factors in non-adipogenic cells is sufficient to convert them to adipocyte-like cells. To determine the requirements of SWI/SNF enzymes in adipocyte differentiation, we introduced PPARγ, C/EBPα or C/EBPβ into fibroblasts that inducibly express dominant-negative versions of the Brahma-Related Gene 1 (BRG1) or human Brahma (BRM), which are the ATPase subunits of the SWI/SNF enzymes. We found that adipogenesis and expression of adipocyte genes were inhibited in the presence of mutant SWI/SNF enzymes. Additionally, in cells expressing C/EBPα or C/EBPβ, PPARγ expression was SWI/SNF dependent. These data indicate the importance of these remodeling enzymes in both early and late gene activation events. Subsequently, we examined by chromatin immunoprecipitation (ChIP) assay the functional role of SWI/SNF enzymes in the activation of PPARγ2, the master regulator of adipogenesis. Temporal analysis of factors binding to the PPARγ2 promoter showed that SWI/SNF enzymes are required to promote preinitiation complex assembly and function. Additionally, our studies concentrated on the role of C/EBP family members in the activation of early and late genes during adipocyte differentiation. During adipogenesis, C/EBPβ and δ are rapidly and transiently expressed and are involved in the expression of PPARγ and C/EBPα, which together activate the majority of the adipocyte genes. Our studies determined the temporal recruitment of the C/EBP family at the promoters of early and late genes by ChIP assay during adipocyte differentiation. We found that all of the C/EBP members evaluated are present at the promoters of early and late genes, and the binding correlated with the kinetics of the C/EBPs expression. Binding of C/EBPβ and δ is transient, subsequently being replaced by C/EBPα. These studies demonstrated that C/EBPβ and δ are not only involved in the regulation of PPARγ and C/EBPα, but also in the activation of late expressed adipocyte genes.

No description available.