Authors: Witte, Anna
Title: Click-mediated enrichment of specific genomic loci
Language (ISO): en
Abstract: In all organisms, the genetic information of cells is stored in the nucleotide sequence of deoxyribonucleic acid (DNA). The human organism consists of more than 200 different somatic cell types with the same genetic information (genotype). Even though, they drastically differ in their morphology and function (phenotype), which is related to different gene expression levels. Gene expression is controlled by macromolecular interactions and epigenetic modifications on chromatin that are highly locus-specific and drive functional aspects of each locus. Even though, the compositions of macromolecules and modifications on many chromosome loci remain poorly understood, in part due to the lack of locus-specific chromatin purification methods that would allow for targeted, discovery-oriented analyses. In this work, the first enrichment method based on bio-orthogonal conjugation (“click-chemistry) with encoded programmable DNA binding domains (transcription-activator like effectors – TALEs) for purification of user-defined genomic loci was established. This click-mediated enrichment provides complementary potential compared to the existing enzymatic biotinylation strategies used in chromatin enrichment methods in the view of site-specificity and proteome-wide background. This method will enable correlations of local chromatin states with phenotypes as the key to a deeper understanding of the regulation landscape of the eukaryotic genome. As a first outlook experiment, we extended our approach to fusion constructs of specific TALE proteins and ten-eleven translocation (TET) dioxygenases for epigenetic editing in vivo. TETs catalyze the oxidation of 5-methylcytosine (5mC) to the oxidized derivates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). In combination with the click-mediated enrichment and proteomics analysis, this will enable studying how local epigenetic changes modulate the local chromatin landscape in vivo as basis for alterations in gene expression.
Subject Headings: Chromatin purification
Genetic code expansion
Subject Headings (RSWK): Epigenetik
Issue Date: 2020
Appears in Collections:Lehrstuhl für Chemische Biologie der Nukleinsäuren

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