Epigenetik Molekularbiologie und Tumorforschung ths Prof. Dr. Suske Guntram Suske, Guntram (Prof. Dr.) ppn:334806496 Publikationsserver der Universitätsbibliothek Marburg Universitätsbibliothek Marburg L3MBTL2 ist ein Mitglied der Familie von MBT-Domänen Proteinen. MBT-Domänen vermitteln die Bindung an methylierte Lysinreste innerhalb der N-Termini von Histonen. L3MBTL2 wurde als transkriptioneller Repressor beschrieben und ist ein Bestandteil verschiedener Multiproteinkomplexe. In Mäusen besitzt L3MBTL2 eine essentielle Funktion für die Embryonalentwicklung und beeinflusst die Proliferation muriner embryonaler Stammzellen. In dieser Arbeit wurde L3MBTL2 als Substrat der SUMOylierung identifiziert. Mutationsanalysen zeigten, dass die spezifische Konjugation von SUMO2/3 an den Lysinresten 675 und 700 im äußersten C-Terminus des Proteins erfolgt. In vitro SUMOylierungs-Experimente identifizierten PIAS1 als E3 Ligase der L3MBTL2 SUMOylierung. Analysen zur molekularen Funktion der SUMOylierung von L3MBTL2 ergaben, dass die SUMOylierung weder die Bindung des Proteins an N-terminale Histonenden, noch dessen Rekrutierung an Chromatin beeinflusst. Mithilfe von ChIP-Seq-Experimenten wurden die genomweiten Bindungsstellen von endogenem L3MBTL2, sowie von überexprimiertem wildtypischem und SUMOylierungs-defizientem L3MBTL2 identifiziert. Endogenes L3MBTL2 wird an Promotoren rekrutiert, die eine aktive Initiation der Transkription aufweisen. Die mit L3MBTL2 in stabilen Multiproteinkomplexen assoziierten Proteine E2F6 und RING2 sowie monoubiquitiniertes H2A wurden ebenfalls an ausgewählten L3MBTL2 Zielpromotoren nachgewiesen. Innerhalb der Bindungssequenzen von L3MBTL2 wurde die E-Box als das am stärksten überrepräsentierte Motiv ermittelt. L3MBTL2 ist mit dem E-Box-bindenden, heterodimeren Transkriptionsfaktor MGA/MAX in stabilen Multiproteinkomplexen assoziiert. Die Rekrutierung L3MBTL2-enthaltender Proteinkomplexe erfolgt daher vermutlich nicht über E2F6, sondern wird wahrscheinlich durch MGA/MAX vermittelt. Mithilfe genomweiter Expressionsanalysen wurden Gene mit einer schwachen L3MBTL2-Rekrutierung als spezifisch durch SUMOyliertes L3MBTL2 reprimierte Zielgene identifiziert. Neben der Analyse der SUMOylierung von L3MBTL2 wurde der Effekt der proteomweiten SUMOylierung auf die Zusammensetzung L3MBTL2-enthaltender Proteinkomplexe untersucht. Dabei führte die Konservierung der proteomweiten SUMOylierung zur Stabilisierung von Multiproteinkomplexen. Die SUMOylierung von L3MBTL2 alleine nahm keinen Einfluss auf die Komplexstabilität. Mithilfe einer massenspektrometrischen Analyse nach Aufreinigung L3MBTL2-enthaltender Komplexe wurden potentielle Interaktionspartner von L3MBTL2 identifiziert. Dabei konnten keine signifikanten Unterschiede zwischen wildtypischem und SUMOylierungs-defizientem L3MBTL2 festgestellt werden. Jedoch wurden infolge der Konservierung der proteomweiten SUMOylierung Proteine wie zum Beispiel GMPS, SUPT16H oder SSRP1 identifiziert, die bislang nicht als Interaktionspartner von L3MBTL2 beschrieben wurden. Viele dieser Proteine stellen selbst Substrate der SUMOylierung dar. Daher kann angenommen werden, dass verschiedene SUMOylierte Komplexkomponenten synergistisch auf die Stabilität eines Komplexes wirken. Die Modifikation eines einzelnen Proteins spielt dagegen eine untergeordnete Rolle. 2013-12-18 urn:nbn:de:hebis:04-z2013-07385 Repressorproteine application/pdf Ubiquitin-ähnliche Proteine SUMOylation doctoralThesis L3MBTL2 https://archiv.ub.uni-marburg.de/diss/z2013/0738/cover.png Transkription Funktionale Analysen zur SUMOylierung des transkriptionellen Repressors L3MBTL2 Medizin Stielow, Christina Stielow Christina German Control of nuclear HIPK2 localization and function by a SUMO interaction motif. Biochimica Et Biophysica Acta 1813, 283–297. 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MBT domains are chromatin-reading modules that mediate binding to methylated lysine residues within histone tails. L3MBTL2 acts as a transcriptional repressor and is associated with several multisubunit complexes. In mice, L3MBTL2 is essential for embryonic development and affects proliferation of murine embryonic stem cells. In this study L3MBTL2 was identified as a substrate for SUMOylation. Mutational analysis demonstrated lysine residues 675 and 700 in the most C-terminal region of the protein to be specifically modified with SUMO2/3. In vitro SUMOylation experiments identified PIAS1 as an E3 ligase of L3MBTL2 SUMOylation. Functional analysis showed that SUMOylation of L3MBTL2 neither affects binding of L3MBTL2 to methylated histone tails nor its recruitment to chromatin. The genome-wide binding sites of endogenous L3MBTL2 as well as overexpressed wild-type and SUMOylation-deficient L3MBTL2 were identified by ChIP-seq. L3MBTL2 is recruited to promoters that possess an active initiation of transcription. E2F6 and RING2, both stably associated with L3MBTL2, as well as monoubiquitinated H2A were also present at selected L3MBTL2 target promoters. The E-box emerged to be the most overrepresented motif within L3MBTL2 binding sequences. L3MBTL2 is stably associated with the heterodimeric E-box-binding transcription factor MGA/MAX. Thus, the recruitment of L3MBTL2-containing complexes presumably does not occur via E2F6, but is likely mediated by MGA/MAX. Genome-wide expression analysis revealed weakly bound L3MBTL2 target genes to be specifically repressed by SUMOylated L3MBTL2. Besides analyzing the role of L3MBTL2 SUMOylation, the effect of proteome-wide SUMOylation on the composition of L3MBTL2-containing complexes was investigated. Conservation of SUMOylation stabilized multisubunit complexes, whereas L3MBTL2 SUMOylation did not affect complex stability. Mass spectrometry of purified L3MBTL2-containing complexes identified potential interaction partners of L3MBTL2 showing no significant differences between wild-type and mutant L3MBTL2-containing complexes. However, due to conserved SUMOylation several proteins like GMPS, SUPT16H or SSRP1, so far not known to be associated with L3MBTL2, were identified with high confidence. Many of these proteins themselves are targets for SUMOylation. Thus, it can be proposed that SUMOylation of several proteins rather than SUMOylation of a single protein affects stability of L3MBTL2-containing complexes. 2013-11-15 https://doi.org/10.17192/z2013.0738 malignant brain tumor 2013 Medical sciences Medicine Medizin