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Engineering Recombinant Protein Sensors for Quantifying Histone Acetylation
journal contribution
posted on 2017-02-08, 00:00 authored by Oscar
F. Sanchez, Agnes Mendonca, Ana D. Carneiro, Chongli YuanH3K14ac
(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.
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Keywords
histone acetylation levelsdetection rangeVarious sensor designshistone modificationsQuantifying Histone Acetylation H 3Kfluorescence intensityhistone modification levelsample pretreatmentsprotein sensormodification levelPBH 3K antibodiesH 3K reader domainepigentic modificationscell extractshistone modificationnovel toolquantifying H 3K levelH 3Kquality antibodiesprobe histone modification changeHEK 293T cellssensor colocalizesAberrant changeslysine 14Current detection techniquesengineering Recombinant Protein Sensorskidney 293 TH 3K levels
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