Programmable 5-Methylcytosine Oxidation and Covalent Capture of Genomic Loci for Targeted Proteomics
Biological and Biomimetic Chemistry
Final Report Abstract
5-Methylcytosine (mC) is a dynamic regulatory element of the mammalian genome that regulates gene expression during embryonic development, cell differentiation and carcinogenesis. mC is introduced into genomes by DNA methyltransferases (DNMT), while the iterative oxidation of mC to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC) and 5-carboxycytosine (caC) by TET dioxygenases initiates active demethylation. Oxidized mC derivatives (oxi-mC) are potential epigenetic markers with unique interactivity for chromatin proteins. Fishing/proteomics studies with synthetic DNAs in nuclear extracts allowed first interaction profiles of oximC. Although these provide valuable first hints on the functions of the base modifications, they do not reflect the complex interaction networks in chromatin and cannot reveal causalities between de novo mC oxidation and changes in the chromatin landscape at selected loci in vivo, such as protein composition and modification. In this project, tools were developed for the enrichment of selected genomic loci from cells for proteomics studies, which can be easily combined with the in situ introduction of base modifications. TALE effectors (TALEs) were designed and expressed in mammalian cell lines. These constructs were equipped with unnatural amino acids (UAA) that enable strain-promoted inverse electron-Diels-Alder cycloaddition (SPIEDAC). After locus-specific binding of TALEs in vivo, formaldehyde cross-linking, DNA isolation and fragmentation, SPIEDAC with tetrazinebiotin conjugates enables isolation of bound chromatin loci by streptavidin-conjugated magnetic solid phases. TALES were designed for binding the repetitive SATIII locus, which is involved in the formation of stress bodies after heat shock. Isolation and MS/MS studies provided new insights into the local changes of the proteome. The established method forms a promising basis for the investigation of local proteome changes by base modifications, for example by fusion of the TALE proteins with DNMT or TET domains.
Publications
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Encoded, click-reactive DNA-binding domains for programmable capture of specific chromatin segments. Chemical Science, 11(46), 12506-12511.
Witte, Anna; Muñoz-López, Álvaro; Metz, Malte; Schweiger, Michal R.; Janning, Petra & Summerer, Daniel
