Development of a BiAD Sensor for Locus-Specific Detection of Cellular Histone Acetylation Dynamics by Fluorescence Microscopy

TL;DR

Researchers developed a new sensor called BiAD to detect changes in histone acetylation in living cells using fluorescence microscopy.

Contribution

The novel BiAD sensor uses a tandem bromodomain to detect locus-specific histone acetylation dynamics in living cells.

Findings

The TTC34 gene body was identified as a suitable target for the BiAD sensor due to its dense histone acetylation and repeated sequences.

A dual-domain bromodomain reader was necessary for effective detection, showing the importance of avidity effects.

The sensor successfully detected increased histone acetylation after treatment with Trichostatin A.

Abstract

Background: Dynamic changes in histone acetylation play crucial roles during cellular differentiation and disease development, but their detection in living cells is still a challenging task. Objectives: Here, we developed a Bimolecular Anchor Detector (BiAD) sensor for the detection of locus-specific changes in histone acetylation in living cells by fluorescence microscopy. Methods: We used the BRD9 bromodomain cloned as tandem double domain (2xBRD9-BD) as a reader of histone acetylation. It was integrated into a dual-color BiAD chassis that was previously described by us. Results: We identified the gene body of TTC34 as a potential target for our sensor, because it contains dense histone acetylation and 392 local sequence repeats. Using a binding-deficient mutant of 2xBRD9-BD as a negative control, we established a successful readout of histone acetylation at the TTC34 locus. A single-domain reader did not function, indicating the requirement for the double reader to enhance the affinity and specificity of the chromatin interaction via avidity effects. With this sensor, we could detect dynamic increases in histone acetylation at the TTC34 locus after the treatment of cells with the histone deacetylase inhibitor Trichostatin A for 6 h indicating the applicability of the sensor for single-cell epigenome studies. Conclusions: Our data demonstrate that active chromatin modifications can be detected by BiAD sensors using 2xBRD9-BD as a reader. This complements the toolkit of the available BiAD sensors and documents the modularity of BiAD sensors.

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