Genetically encoded FRET fluorescent sensor designed for detecting MOF histone acetyltransferase activity in vitro and in living cells

Acetylation of lysine in the histone H4 N-terminal is one of the most significant epigenetic modifications in cells. Aberrant changes involving lysine acetylation modification are commonly reported in multiple types of cancers. Currently, whether it is for in vivo or in vitro, there are limited approaches for the detection of H4 lysine acetylation levels. In particular, the main problems are the high cost and the cumbersome detection process, such as for radioactive C-14 isotope detection. Therefore, there is an important need to develop a simple, fast, and low-cost means of detection. In this study, we reported the development of a gene-coding protein sensor. This protein sensor was designed based on the mechanism of fluorescence resonance energy transfer (FRET). The four kinds of sensors, varying from substrate and linker length, were evaluated, with similar to 20% increases in response efficiency. Next, sensors with different lysine mutation sites in the substrate sequence or mutation of key amino acids in the binding domain were also analyzed to determine site specificity. We found single-site lysine mutant could not cause a significant decrease in response efficiency. Furthermore, addition of MG149, a histone acetyltransferase inhibitor, resulted in a decrease in the ratio change value. Moreover, histone deacetylase1 HDAC1 was also found to reduce the ratio change values when added to reaction system. Finally, the optimized sensor was applied to living cells and established to provide a sensitive response with overexpression and knockdown of MOF (males absent on the first). These results indicated that the sensor can be used for screening chemical drugs regulating H4 N-terminal lysine acetylation level in vitro, as well as monitoring dynamic changes of lysine acetylation levels in living cells.

Automated summary

This study developed a gene-coding protein sensor based on the mechanism of fluorescence resonance energy transfer for detecting H4 lysine acetylation levels. Different sensors with varying substrate sequences were found to increase response efficiency, while single-site lysine mutation did not significantly decrease efficiency. Inhibitors and histone deacetylase added to the system showed effects on the sensor's response.