Die Rolle des Transkriptionsfaktors AP-2γ in primordialen Keimzellen

In der vorgelegten Arbeit sollte sowohl die Expression von AP-2γ in frühen Stadien primordialer Keimzellen, als auch die Konsequenzen des Verlustes von AP-2γ auf die molekularen Mechanismen in primordialen Keimzellen mittels geeigneter in vitro Systeme untersucht werden. AP-2γ gehört zu der Familie der AP-2-Transkriptionsfaktoren, die jeweils aus Mitgliedern bei Mensch und Maus besteht. Diese Transkriptionsfaktoren werden während der Embryogenese in verschiedenen Geweben exprimiert und werden mit Differenzierung, Zellzykluskontrolle, Proliferation und Apoptose in Verbindung gebracht. Zudem haben frühere Arbeiten gezeigt, dass der Verlust von AP-2γ im Embryo zu einem Keimzellverlust kurz nach der Spezifizierung führt, sodass die Frage aufkommt, welche Rolle AP-2γ in der Keimzellbiologie spielt. Primordiale Keimzellen werden früh während der Embryogenese in der Basis der Allantois etabliert (E7.25-7.5) und beginnen anschließend mit der Migration durch den Embryo zu den Genitalanlagen. Bereits während der Spezifizierung spielen komplexe genetische Vorgänge eine sehr wichtige Rolle. Dabei ist das Protein Blimp-1 essentiell, da es sowohl keimzellspezifische Gene aktiviert als auch Gene reprimiert, die für eine somatische Differenzierung verantwortlich sind, und somit die primordialen Keimzellen von den umgebenden Zellen abgrenzt. In dieser Arbeit konnte die Expression von AP-2γ in primordialen Keimzellen in den Stadien E7.5-8.5 gezeigt werden. Zudem konnte gezeigt werden, dass dieser Verlust der Keimzellen in den Nullmutanten nicht durch Apoptose verursacht wird. Um die molekularen Mechanismen, die diesem Keimzellverlust unterliegen, zu untersuchen, wurden zwei in vitro Systeme etabliert. Dazu wurden zum einen murine embryonale Stammzellen (AP-2γ+/+ und AP-2γ-/-) in EB-Kulturen zu primordialen Keimzellen differenziert, des Weiteren wurde die humane Keimzelltumorlinie TCam-2 als Seminoom-Zelllinie charakterisiert und somit als geeignetes in vitro System für Keimzellen etabliert.In vitro konnte gezeigt werden, dass der Verlust von AP-2γ zu einer Hochregulation von mesodermalen Markern sowohl im murinen, als auch im humanen System führt. Zudem geht der Verlust von AP-2γ mit einer Deregulation von miRNS-Molekülen einher, die mit mesodermaler Differenzierung in Verbindung gebracht werden. Den Ergebnissen dieser Arbeit nach scheint AP-2γ in einer Kaskade unterhalb von Blimp1 zu liegen und verhindert die Differenzierung primordialer Keimzellen in mesodermale Zellen

The AP-2 family of transcription factors

The AP-2 family of transcription factors consists of five different proteins in humans and mice: AP-2α, AP-2β, AP-2γ, AP-2δ and AP-2ε. Frogs and fish have known orthologs of some but not all of these proteins, and homologs of the family are also found in protochordates, insects and nematodes. The proteins have a characteristic helix-span-helix motif at the carboxyl terminus, which, together with a central basic region, mediates dimerization and DNA binding. The amino terminus contains the transactivation domain. AP-2 proteins are first expressed in primitive ectoderm of invertebrates and vertebrates; in vertebrates, they are also expressed in the emerging neural-crest cells, and AP-2α(-/- )animals have impairments in neural-crest-derived facial structures. AP-2β is indispensable for kidney development and AP-2γ is necessary for the formation of trophectoderm cells shortly after implantation; AP-2α and AP-2γ levels are elevated in human mammary carcinoma and seminoma. The general functions of the family appear to be the cell-type-specific stimulation of proliferation and the suppression of terminal differentiation during embryonic development

Critical function of AP-2gamma/TCFAP2C in mouse embryonic germ cell maintenance

Formation of the germ cell lineage involves multiple processes, including repression of somatic differentiation and reacquisition of pluripotency as well as a unique epigenetic constitution. The transcriptional regulator Prdm1 has been identified as a main coordinator of this process, controlling epigenetic modification and gene expression. Here we report on the expression pattern of the transcription factor Tcfap2c, a putative downstream target of Prdm1, during normal mouse embryogenesis and the consequences of its specific loss in primordial germ cells (PGCs) and their derivatives. Tcfap2c is expressed in PGCs from Embryonic Day 7.25 (E 7.25) up to E 12.5, and targeted disruption resulted in sterile animals, both male and female. In the mutant animals, PGCs were specified but were lost around E 8.0. PGCs generated in vitro from embryonic stem cells lacking TCFAP2C displayed induction of Prdm1 and Dppa3. Upregulation of Hoxa1, Hoxb1, and T together with lack of expression of germ cell markers such Nanos3, Dazl, and Mutyh suggested that the somatic gene program is induced in TCFAP2C-deficient PGCs. Repression of TCFAP2C in TCam-2, a human PGC-resembling seminoma cell line, resulted in specific upregulation of HOXA1, HOXB1, MYOD1, and HAND1, indicative of mesodermal differentiation. Expression of genes indicative of ectodermal, endodermal, or extraembryonic differentiation, as well as the finding of no change to epigenetic modifications, suggested control by other factors. Our results implicate Tcfap2c as an important effector of Prdml activity that is required for PGC maintenance, most likely mediating Prdm1-induced suppression of mesodermal differentiation

Interference with Activator Protein-2 transcription factors leads to induction of apoptosis and an increase in chemo- and radiation-sensitivity in breast cancer cells

<p>Abstract</p> <p>Background</p> <p>Activator Protein-2 (AP-2) transcription factors are critically involved in a variety of fundamental cellular processes such as proliferation, differentiation and apoptosis and have also been implicated in carcinogenesis. Expression of the family members AP-2α and AP-2γ is particularly well documented in malignancies of the female breast. Despite increasing evaluation of single AP-2 isoforms in mammary tumors the functional role of concerted expression of multiple AP-2 isoforms in breast cancer remains to be elucidated. AP-2 proteins can form homo- or heterodimers, and there is growing evidence that the net effect whether a cell will proliferate, undergo apoptosis or differentiate is partly dependent on the balance between different AP-2 isoforms.</p> <p>Methods</p> <p>We simultaneously interfered with all AP-2 isoforms expressed in ErbB-2-positive murine N202.1A breast cancer cells by conditionally over-expressing a dominant-negative AP-2 mutant.</p> <p>Results</p> <p>We show that interference with AP-2 protein function lead to reduced cell number, induced apoptosis and increased chemo- and radiation-sensitivity. Analysis of global gene expression changes upon interference with AP-2 proteins identified 139 modulated genes (90 up-regulated, 49 down-regulated) compared with control cells. Gene Ontology (GO) investigations for these genes revealed <it>Cell Death </it>and <it>Cell Adhesion and Migration </it>as the main functional categories including 25 and 12 genes, respectively. By using information obtained from Ingenuity Pathway Analysis Systems we were able to present proven or potential connections between AP-2 regulated genes involved in cell death and response to chemo- and radiation therapy, (i.e. <it>Ctgf, Nrp1</it>, <it>Tnfaip3, Gsta3</it>) and AP-2 and other main apoptosis players and to create a unique network.</p> <p>Conclusions</p> <p>Expression of AP-2 transcription factors in breast cancer cells supports proliferation and contributes to chemo- and radiation-resistance of tumor cells by impairing the ability to induce apoptosis. Therefore, interference with AP-2 function could increase the sensitivity of tumor cells towards therapeutic intervention.</p

Evolution of the eukaryotic ARP2/3 activators of the WASP family: WASP, WAVE, WASH, and WHAMM, and the proposed new family members WAWH and WAML

<p>Abstract</p> <p>Background</p> <p>WASP family proteins stimulate the actin-nucleating activity of the ARP2/3 complex. They include members of the well-known WASP and WAVE/Scar proteins, and the recently identified WASH and WHAMM proteins. WASP family proteins contain family specific N-terminal domains followed by proline-rich regions and C-terminal VCA domains that harbour the ARP2/3-activating regions.</p> <p>Results</p> <p>To reveal the evolution of ARP2/3 activation by WASP family proteins we performed a "holistic" analysis by manually assembling and annotating all homologs in most of the eukaryotic genomes available. We have identified two new families: the WAML proteins (WASP and MIM like), which combine the membrane-deforming and actin bundling functions of the IMD domains with the ARP2/3-activating VCA regions, and the WAWH protein (WASP without WH1 domain) that have been identified in amoebae, Apusozoa, and the anole lizard. Surprisingly, with one exception we did not identify any alternative splice forms for WASP family proteins, which is in strong contrast to other actin-binding proteins like Ena/VASP, MIM, or NHS proteins that share domains with WASP proteins.</p> <p>Conclusions</p> <p>Our analysis showed that the last common ancestor of the eukaryotes must have contained a homolog of WASP, WAVE, and WASH. Specific families have subsequently been lost in many taxa like the WASPs in plants, algae, Stramenopiles, and Euglenozoa, and the WASH proteins in fungi. The WHAMM proteins are metazoa specific and have most probably been invented by the Eumetazoa. The diversity of WASP family proteins has strongly been increased by many species- and taxon-specific gene duplications and multimerisations. All data is freely accessible via <url>http://www.cymobase.org</url>.</p