Imaging-based analysis of 5-methylcytosine at low-repetitive genomic loci using transcription activator-like effector probes
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Date
2023
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Abstract
5-Methylcytosine (5mC) is the main epigenetic modification of mammalian genomes. It plays significant roles during cell development and differentiation and is involved in the regulation of essential cellular processes such as the control of gene expression. Dysregulation of methylation can lead to aberrant epigenetic patterns associated with a variety of diseases. To analyze cellular 5mC in situ, fluorescently labeled transcription-activator-like effector (TALE) proteins can be used as 5mC-sensitive probes in imaging studies. TALEs are DNA-binding proteins that provide sequence and 5mC selectivity via a domain of modular repeats, each recognizing a specific nucleobase. This enables the design of TALE probes for sequence-specific analysis of 5mC in user-defined target sequences. In imaging studies, 5mC-sensitive and 5mC-insensitive TALE pairs are used in co-stainings to allow the analysis of 5mC independently of changes in target accessibility. However, until now this has been limited to highly repetitive genomic DNA sequences. To extend this approach for the analysis of 5mC in low-repetitive coding gene loci, this work develops a straightforward signal amplification strategy to increase the imaging sensitivity with TALEs. This is achieved by additional immunostaining of the employed TALE probes, enabling the imaging of only 32 theoretical repeat sequences in the low repetitive MUC4 gene locus. In co-staining experiments, this allows the detection of 5mC changes in this locus between cell types with different methylation levels, introduced by DNA methyltransferase knockouts or overexpression. The ability to detect 5mC differences in this small number of target sequences opens up new perspectives for the analysis of 5mC in non-repetitive genomic loci, providing new insights into the regulation of gene expression.
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Epigenetik, DNA-Methylierung, Fluoreszenzmikroskopie, MUC4