Funktionale Analysen zur SUMOylierung des transkriptionellen Repressors L3MBTL2

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...

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Bibliographic Details
Main Author: Stielow, Christina
Contributors: Suske, Guntram (Prof. Dr.) (Thesis advisor)
Format: Dissertation
Language:German
Published: Philipps-Universität Marburg 2013
Molekularbiologie und Tumorforschung
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Table of Contents: L3MBTL2 belongs to the family of MBT domain proteins. 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.