Engineering Recombinant Protein Sensors for Quantifying Histone Acetylation
2017-02-08T00:00:00Z (GMT) by
H3K14ac (acetylation of lysine 14 of histone H3) is one of the most important epigentic modifications. Aberrant changes in H3K14ac have been associated with various diseases, including cancers and neurological disorders. Tools that enable detection and quantification of H3K14ac levels in cell extracts and in situ are thus of critical importance to reveal its role in various biological processes. Current detection techniques of specific histone modifications, however, are constrained by tedious sample pretreatments, lack of quantitative accuracy, and reliance on high quality antibodies. To address this issue, we engineered recombinant sensors that are suitable for probing histone acetylation levels using various biological samples. The protein sensor contains recongition domain(s) with sequences derived from the bromodomain of human polybromo-1 (PB1), a natural H3K14ac reader domain. Various sensor designs were tested using nuclear extracts and live cells. The sensor containing dimeric repeats of bromodomain was found most effective in quantifying H3K14ac level in both in vitro and in situ assays. The sensor has a linear detection range of 0.5–50 nM when mixed with nuclear extracts. The sensor colocalizes with H3K14ac antibodies in situ when transfected into human embryonic kidney 293T (HEK293T) cells and is thus capable of providing spatial details of histone modification within the nucleus. Corrected nuclear fluorescence intensity was used to quantify the modification level in situ and found to correlate well with our in vitro assays. Our sensor offers a novel tool to characterize the histone modification level using nuclear extracts and probe histone modification change in live cells.