Functional characterisation of LINT and other dCoREST complexes

果蝇的l(3)mbt基因突变可导致恶性脑肿瘤的形成。为了更好地了解L(3)mbt蛋白的分子功能，我们纯化了L(3)mbt相关多肽，并鉴定了含L(3)mbt的LINT复合物。LINT复合物是由三个亚基（L(3)mbt， LINT -1和CoREST）组成的分子机器。它与染色质结合，抑制脑肿瘤相关基因的转录。我们已经确定了该复合物的基因组结合位点，确定了其靶基因，并建立了检测方法来研究其分子抑制机制。在LINT的纯化过程中，我们发现了额外的复合物，这些复合物也含有CoREST亚基。在第二个资助期内，我们希望解决有关LINT复合体的重要问题，并希望对新发现的CoREST蛋白复合体家族进行功能表征。我们将从重组亚基重建LINT，定义对复杂完整性重要的结构域，并确定LINT与修饰的多核小体在体外的相互作用。此外，我们将定义允许LINT特异性结合其靶基因的参数，并阐明其基因抑制机制。我们将以果蝇的翅膀为模型，分析LINT在细胞命运规范中的作用。此外，我们将通过系统地纯化和表征所有含有CoREST的蛋白复合物，来表征果蝇的新型CoREST蛋白复合物家族。鉴于L(3)mbt和CoREST在人类中都是保守的，我们希望我们的研究能够揭示与疾病相关的分子机制。

Mutation of the l(3)mbt gene of Drosophila cause the formation of malignant brain tumours. In order to better understand the molecular functions of L(3)mbt protein, we have purified L(3)mbt-associated polypeptides and identified the L(3)mbt-containing LINT complex. The LINT complex is a molecular machine that consists of three subunits (L(3)mbt, Lint-1 and CoREST). It binds to chromatin and represses the transcription of brain tumour-associated genes. We have determined the genomic binding sites of the complex, have defined its target genes and established assays to investigate its molecular repression mechanisms. During purification of LINT we have discovered additional complexes which also contain a CoREST subunit.During the second funding period we want to address important questions about the LINT complex and we want to functionally characterise the newly discovered CoREST protein complex family. We will reconstitute LINT from recombinant subunits, define domains that are important for complex integrity and determine the interactions between LINT and modified polynucleosomes in vitro. In addition, we will define the parameters that allow LINT to specifically bind to its target genes and we will elucidate its mechanism of gene repression. We will analyse the role of LINT in cell fate specification using the Drosophila wing as a model. In addition, we will characterise the novel CoREST protein complex familiy of Drosophila, by systematically purifying and characterising all CoREST-containing protein complexes.Given that both L(3)mbt and CoREST are conserved in humans, we hope that our studies will shed light on disease-relevant molecular mechanisms.