Programmable 5-Methylcytosine Oxidation and Covalent Capture of Genomic Loci for Targeted Proteomics

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Mammalian gene expression is dynamically regulated by genomic 5-methylcytosine (mC). mC is introduced by DNA methyltransferases, whereas iterative oxidation of mC to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC) by ten-eleven translocation (TET) dioxygenases controls active demethylation. These oxidized mC derivatives (oxi-mC) are potential epigenetic markers in their own right, with unique interaction abilities to chromatin proteins. Initial fishing/proteomics studies with synthetic DNAs in nuclear extracts have provided first interaction profiles of hmC, fC and caC. However, though giving invaluable initial clues to the functions of the bases, such experiments do not reflect the complex interaction networks of chromatin, and cannot establish causalities between de novo mC oxidation and changes in the landscape of natural chromatin at user-defined loci in vivo, such as protein composition and protein modifications. In this project, we will develop tools for the intracellular mC oxidation at user-defined genomic loci combined with the covalent capture of these loci for proteomics studies. This will enable 1.) the discovery of proteins that are directly or indirectly recruited or repelled, and 2.) enable the discovery of what histone and other protein posttranslational modifiations may be written or erased by mC oxidiation. We will design fusion constructs of TET enzymes and transcription-activator-like effectors (TALEs) and express them in mammalian cell lines for oxidiation. We will equip these constructs with cyclooctyne handles for strain-promoted inverse electron Diels-Alder cycloaddions (SPIEDAC) using genetic code expansion. After locus-specific binding and mC oxidiation in vivo, formaldehyde crosslinking of the construct and other chromatin proteins followed by DNA isolation and fragmentation, SPIEDAC with tetrazine-functionalized beads will enable the fully covalent capture of the bound chromatin loci for proteomic analyses. Covalent capture by SPIEDAC should overcome limitations of noncovalent capture approaches in view of sensitivity, selectivity and ability for stringent removal of false-positives. We will first apply this approach to the repetitive SATIII locus involved in stress body formation after heat shock, to study epigenetic control mechanism of this process. Afterwards, we will target the three single gene loci CDKN2A, BRCA1 and ZAP70 involved in mC-controlled cancer development in different cell types that cover different promoter types in respect to CpG densities. From these studies, we anticipate new insights into how oxi-mC are involved in regulatory, direct and indirect protein recruitment and release and how they are integrated in the crosstalk between DNA and protein (histone) modifications. Our approach provides a new avenue to the discovery of readers of oxi-mC by identifying such interactions in natural chromatin and in different locus- and cellular contexts.