Dokument

Summary:
The Drosophila Lethal (3) malignant brain tumour (dL(3)mbt) protein is the founding member of the family of MBT domain proteins. The MBT domain is a ‘chromatin reader’, a module that specifically recognises mono- and di-methylated lysines within histone tails. In vitro studies suggest that these domains compact nucleosomes to form higher order chromatin structures. Importantly, MBT domain-containing proteins were previously shown to have critical functions in developmental processes, maintenance of transcriptional repression and tumour suppression. Accordingly, mutation of the gene that encodes the dL(3)mbt protein leads to the development of a malignant and invasive tumour in the brain of third instar larvae. Data from both Drosophila melanogaster and human implicate that MBT domain proteins cooperate with or are part of multi-subunit protein complexes. Therefore, the goal of this thesis was to identify novel interaction partners and protein complexes of dL(3)mbt. In the first part of this thesis, dL(3)mbt has been shown to associate with enzymatic histone deacetylase activity. dRpd3 was identified as a histone deacetylase specifically associating with dL(3)mbt. Interestingly, the three MBT domains were sufficient to mediate interaction with dRpd3. This dL(3)mbt-dRpd3 complex has been linked to the maturation of newly synthesised chromatin. In the second part of this study, the multi-subunit complex LINT, consisting of dL(3)mbt, the co-repressor protein dCoREST and the novel protein dLint-1, was isolated by FLAG immunoaffinity purification and classical biochemical chromato-graphy. These factors interacted stably with each other in extracts from cell lines, embryos and larval brain. On polytene chromosomes the two LINT subunits dL(3)mbt and dLint-1 co-localised extensively at many binding sites. Subsequent microarray analysis led to the identification of hundreds of genes that were co-regulated by dL(3)mbt and dLint-1. Among these target genes was a subset of germline-specific genes that were stably repressed by all three LINT complex components. Strikingly, there is a significant overlap with genes that have been demonstrated to drive tumour growth in l(3)mbt mutant brains. Moreover, this study confirmed that the LINT complex bound directly to promoter regions of these target genes to repress transcription. A reporter gene assay revealed that the recruitment of dL(3)mbt and dLint-1 to the promoter of a luciferase gene was sufficient to repress its transcription and that maximal repression was dependent of the presence on all LINT subunits. The upregulation of LINT target genes was accompanied by changes in histone modifications, namely an increase in active and a loss of repressive histone modifications. However, since the LINT complex itself does not contain histone-modifying enzymes these changes are likely to occur co-transcriptionally. Collectively, the results of this thesis lead to a model, in which the LINT complex maintains transcriptional repression of germ cell-specific target genes by hindering access of RNA Polymerase II and other activating transcription factors to their promoters.

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