Abstract
Exploring spatial organization and relationship of diverse biomolecules within cellular nanoenvironments is important to elucidate the fundamental processes of life. However, it remains methodologically challenging. Herein, we report a molecular recognition mechanism cellular macromolecules-tethered DNA walking indexing (Cell-TALKING) to probe the nanoenvironments containing diverse chromatin modifications. As an example, we characterize the nanoenvironments of three DNA modifications around one histone posttranslational modification (PTM). These DNA modifications in fixed cells are labeled with respective DNA barcoding probes, and then the PTM site is tethered with a DNA walking probe. Cell-TALKING can continuously produce cleavage records of any barcoding probes nearby the walking probe. New 3’-OH ends are generated on the cleaved barcoding probes to induce DNA amplification for downstream detections. Combining fluorescence imaging, we identify various combinatorial chromatin modifications and investigate their dynamic changes during cell cycles. We also explore the nanoenvironments in different cancer cell lines and clinical specimens. In principle, using high-throughput sequencing instead of fluorescence imaging may allow the detection of complex cellular nanoenvironments containing tens of biomolecules such as transcription factors.
Highlights
Exploring spatial organization and relationship of diverse biomolecules within cellular nanoenvironments is important to elucidate the fundamental processes of life
We have demonstrated that Cell-TALKING can probe cellular nanoenvironments containing diverse biomolecules
In the context of chromatin, we have explored the nanoenvironments containing three DNA modifications around histone posttranslational modification (PTM)
Summary
Exploring spatial organization and relationship of diverse biomolecules within cellular nanoenvironments is important to elucidate the fundamental processes of life. Chromatin is made up of basic structural subunits called nucleosomes (~10 nm) and is extensively decorated by diverse DNA modifications and histone posttranslational modifications (PTMs)[1] These chemical modifications can be recognized by specialized proteins or protein complexes. We here define many DNA modifications around individual histone PTM as one type of nanoenvironments of chromatin modifications It may reveal the potential interactions between their reader proteins in a spatiotemporal manner, and help elucidate complex epigenetic function in cellular events and pathological processes. Different sequencing strategies have been developed for genome-wide profiling of DNA modifications including 5mC, 5hmC, 5fC, or 5hmU4–7,9,16 These methods reported chemical or chemoenzymatic labeling of modified bases, and revealed their important biological functions. This method is limited to 5mC only and is unable to assess other DNA modifications
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