Novel interaction partners of the chromatin remodeler CHD7, a protein mutated in CHARGE syndrome

Summary CHARGE syndrome, an autosomal dominant inherited multiple malformation syndrome is caused by mutations in the chromodomain helicase DNA binding protein 7 gene (CHD7). In 10% of ‘typical’ CHARGE patients and 40-50% of ‘atypical’ CHARGE patients no mutation in the CHD7 gene is detectable and therefore the molecular cause is still unknown. CHD7 is a nuclear chromatin remodeling protein found in big protein complexes and regulating gene expression of various genes. We suggested that the characterization of CHD7 interacting partners can be useful for understanding the pathogenesis of CHARGE syndrome. Furthermore, for other genetic diseases it was shown that mutations in interacting partners lead to the same or a similar phenotype. Therefore, we hypothesized that CHD7 interacting partners are good candidates, leading to CHARGE syndrome when they are mutated. In the first part of the thesis, CHD8 was identified as an interacting partner of CHD7. The interaction of the CHD7 part (1593 – 2178aa) with the CHD8 part (1789 – 2302aa) was validated by different molecular techniques. Additionally, we could show that the interaction takes place in the nucleoplasm. Furthermore, the influence of 3 known (p.His2096Arg, p.Val2102Ile and p.Gly2108Arg) and one (p.Trp2091Arg) newly identified CHD7 missense mutation on the CHD7-CHD8 interaction was elucidated by direct Yeast two hybrid (Y2H) and co-immunoprecipitation (Co-IP) methods. By Y2H we could demonstrate that the missense mutations p.Trp2091Arg, p.His2096Arg and p.Gly2108Arg disrupt the CHD7-CHD8 interaction, while no influence on the interaction could be seen in Co-IP assasys. Therefore, we suggest that CHD7 and CHD8 interacting directly (shown by Y2H) and indirectly via additional linker proteins which build together with CHD7 and CHD8 a large protein complex (explaining the Co-IP result). In addition, we reported about the sequence analysis of the CHD8 gene in CHD7 mutation negative patients with CHARGE syndrome. In the second part of the thesis, we showed the results of a SILAC and mass-spectrometry approach that identified the hitherto unknown protein FAM124B as a member of CHD7-CHD8 containing complex. The interaction with the CHD8 part (1789 – 2302aa) was validated by direct Y2H and Co-IP methods whereas the interaction with the CHD7 part (1593 – 2178aa) could be only confirmed with the Co-IP assay, demonstrating that FAM124B interacts not directly with the analyzed CHD7 region. Furthermore, we characterized FAM124B. We could show that FAM124B is localized in the nucleoplasm like CHD7 and CHD8. Murine Fam124B is expressed in the organs affected in CHARGE syndrome. High expression could be observed in the developing brain, heart, lung and spinal cord of E12.5 mouse embryos. Recently, CHD8 mutations are shown to be a cause of autism spectrum disorders (ASD) as well as neurodevelopmental disorders (NDD). In conclusion, our results indicate that the hitherto uncharacterized protein FAM124B might be very important for embryonic development and could be involved in the pathogenesis of CHARGE syndrome and ASD/NDDs. Moreover, to analyse the possible role of FAM124B in the pathogenesis of CHARGE and neurocristopathies, we plan to perform a knockdown of FAM124B in Xenopus laevis and test its influence on genes related to neural crest formation. Generation of a knockout mouse model for Fam124B and further characterization of the CHD7 interacting complex members will help to learn more about the molecular mechanism behind CHARGE syndrome and ASD/NDDs.