Research Report 2010 - MDC

Structure of the GroupHarald SaumweberGroup LeaderProf. Dr. HaraldSaumweberScientistsDr. Dereje NegeriPhD studentsDr. Miao GanShaza Dehne*UndergraduatesAndrea Kiep*Nadine Schweizer*Alexander Glahs**part of the period reportedNuclear Signaling andChromosomal DomainsUsing Drosophila as a model our group investigates chromatin switches that arecrucial for Notch and TGF-β signal transduction and mechanisms involved incompartmentalization of chromatin.Skip/Bx42 dependent target gene and cell cycleregulation related to Notch signalingOn binding transmembrane ligands of adjacent cellsthe Notch receptor splits off its intracellular domain(Notch-IC) which migrates into the nucleus and transientlybinds to its target genes. On binding Notchrecruits an activator complex to genes that previouslywere repressed by a silencing complex bound to a chromatinplatform provided by the conserved CSL proteinsCBF1/Su(H)/Lag3. By exchanging the repressor complexfor the Notch activator complex target genes becomeactivated. However, neither the exact composition ofthe activator and the repressor complexes nor themode of the switching mechanism are as yet known.We previously characterized Bx42/SKIP a conservedchromatin co-regulator protein. As in Drosophila themammalian homologue interacts with nuclear componentsof the Notch pathway like Notch-IC andSu(H)/CBF1. By induced Bx42-RNAi we demonstratedthat these interactions are biologically important sincethe expression of several Notch target genes is dependentof the presence of Bx42/SKIP and tissue specificknock down of Bx42/SKIP results in Notch-like phenotypes.As a precondition for crystallographic studies wecurrently map the interaction sites of Bx42/SKIP withNotch-IC, Su(H) and the Hairless protein. For instance, incontrast to previous work by others we mapped theSkip interaction side on the C-term of Notch-IC downstreamof the ankyrin repeats and the RAM domain.Work is in progress to investigate how the Skip/Notch-IC interaction is translated into the activation of targetgenes Expression of the most conserved part of Skip,the central SNW region, results in dominant negativephenotypes that are suppressed by over-expression ofthe FL Skip protein, by Notch gain of function mutationsor putative Skip interacting proteins and enhanced byNotch loss of function alleles. Over-expression of SNWregion in late larval eye discs results in a small eye phenotypeby specific suppression of the Notch-dependentdivision of retinal precursor cells at the G2-M transitionthat is cyclin A dependent. Comparing the expressionprofile of the Bx42 dominant negative to wild type cellson microarrays we find that a restricted number ofgenes is affected by SNW over-expression, amongstthem Dp, a dimerization partner of E2F and the chromatinrepressor protein Sina. Interestingly, in mammaliancells Bx42/SKIP interacts with and counteractsthe repressive effect of the Rb protein and shows aninteraction with the E2F family of cell cycle regulators.The role of Skip and interacting proteins in cell cycleregulation will be investigated in more detail inDrosophila cell culturesChromatin domains and boundariesTransgenes inserted into the mammalian genome regularlybecome silenced and occasionally hyperactivatedat their site of insertion reflecting the impact of localchromatin structure. Interestingly, elements found atchromosomal domain boundaries shield for sucheffects by providing enhancer blockers or barrier ele-106 Cancer Research