Chromatin organization is crucial for gene regulation and as such undergoes large-scale dynamic shifts during a variety of biological processes. However, existing methods typically capture only present snapshots of chromatin architecture. To track these temporal changes, we are developing an in vivo system that enables retrospective chromatin profiling, providing a window into past chromatin states.
To this end, we generated a transgenic mouse line with DOX-inducible, bicistronic expression of a DNA adenine methylase (Dam). Dam deposits 6-methyladenine (6mA) – a modification nearly absent in mammalian genomes – preferentially at accessible GATC motifs in open chromatin regions. Time-controlled induction creates a record of past chromatin landscapes, while expression via rare ribosomal reinitiation events is designed to limit Dam concentration and to reduce background noise. To complement the in vivo model, we aim to develop an adapted library preparation protocol that combines tagmentation-based fragmentation and methylation-sensitive restriction enzymes to enrich for 6mA-labeled fragments.
We successfully introduced the system into mouse embryonic stem cells and confirmed DOX-inducible expression in vitro. Our initial library preparation method generated high-quality sequencing data that correlated well with ATAC-seq profiles from matched cell populations, validating that Dam-labeled regions correspond to accessible chromatin. Importantly, we established a viable transgenic mouse line, in which the system shows no noticeable detrimental effects on animal health or development, demonstrating the compatibility of this approach for in vivo studies. Building on this, we are currently refining the library preparation protocol to further reduce input requirements and to enable diverse applications in vivo.
With this project, we aim to expand the available toolkit for studying chromatin dynamics by enabling retrospective chromatin profiling in a mammalian model organism. The approach opens new potential avenues for understanding how chromatin states change over time in physiologically relevant contexts including development and disease.