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. 2015 Jan 20;87(2):892-9.
doi: 10.1021/ac502629r. Epub 2014 Dec 24.

Genetically encoded molecular biosensors to image histone methylation in living animals

Thillai V Sekar  1 , Kira Foygel, Juri G Gelovani, Ramasamy Paulmurugan
Affiliations

Genetically encoded molecular biosensors to image histone methylation in living animals

Thillai V Sekar et al. Anal Chem. .

Abstract

Post-translational addition of methyl groups to the amino terminal tails of histone proteins regulates cellular gene expression at various stages of development and the pathogenesis of cellular diseases, including cancer. Several enzymes that modulate these post-translational modifications of histones are promising targets for development of small molecule drugs. However, there is no promising real-time histone methylation detection tool currently available to screen and validate potential small molecule histone methylation modulators in small animal models. With this in mind, we developed genetically encoded molecular biosensors based on the split-enzyme complementation approach for in vitro and in vivo imaging of lysine 9 (H3-K9 sensor) and lysine 27 (H3-K27 sensor) methylation marks of histone 3. These methylation sensors were validated in vitro in HEK293T, HepG2, and HeLa cells. The efficiency of the histone methylation sensor was assessed by employing methyltransferase inhibitors (Bix01294 and UNC0638), demethylase inhibitor (JIB-04), and siRNA silencing at the endogenous histone K9-methyltransferase enzyme level. Furthermore, noninvasive bioluminescence imaging of histone methylation sensors confirmed the potential of these sensors in monitoring histone methylation status in response to histone methyltransferase inhibitors in living animals. Experimental results confirmed that the developed H3-K9 and H3-K27 sensors are specific and sensitive to image the drug-induced histone methylation changes in living animals. These novel histone methylation sensors can facilitate the in vitro screening and in vivo characterization of new histone methyltransferase inhibitors and accelerate the pace of introduction of epigenetic therapies into the clinic.

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Figures

Figure 1
Figure 1
Schematic illustration of the concept and the design of histone methylation imaging sensor.
Figure 2
Figure 2
Optimization of split-RLuc fragments. (A) RLuc signal measured from HEK293T-cells transfected with complementation sensors constructed with HP1 and K9-interacting partners with N- and C-terminal RLuc fragments from humanized (NhRL and ChRL) or red-shifted mutant RLuc (NhRL8.6 and ChRL8.6). (B) Optimal number of H3–K9-peptide and the chromodomain needed to achieve efficient sensor signal: RLuc signal measured from HEK293T-cells transfected with complementation sensors constructed with K9 peptide from H3 protein and interacting chromodomain from Suv39H1, in various copy numbers tested for sensors efficiency in measuring histone methylation. (C) Immunoblot analysis of H3–K9-sensor methylation detected by methylation specific antibody: The upper panel shows the wild-type and mutant sensor proteins detected from the immunoprecipitated samples by H3–K9 dimethyl antibody, and the lower panel shows the endogenous H3–K9 proteins detected from the cell lysates of respective samples by the same antibody. (D) H3–K9-sensor methylation detected by methylation specific antibodies after immunoprecipitation: The upper panel shows the wild-type and mutant sensor proteins detected with H3–K9 dimethyl antibody, and the lower panel shows the sensor proteins detected by FLAG antibody. (E) Fluorescence images show the localization of NLS-bearing methylation sensor tagged with EGFP-fusion in the nucleus. (F) Upper panel shows RLuc signal measured from HEK293T cells transfected with complementation sensor-EGFP-fusions with and without NLS shown in (E). Lower panel shows the immunoblot analysis of cell lysates of respective samples in graph using EGFP antibody. GAPDH expression was used to normalize the results. In all experiments, the constructs with RLuc reporter fragments and the interacting protein fragments are in the order they appear in the X-axis labels.
Figure 3
Figure 3
Specificity of histone methylation sensors. (A) RLuc signal measured from HEK293T cells transfected with H3–K9 wild-type and mutant complementation sensors. (B) RLuc signal measured from HEK293T cells transfected with H3–K27 and H3–L27 sensors with no NLS. (C) RLuc signal measured from HEK293T cells transfected with H3–K9 wild-type and Suv39H1 mutant (tryptophan at amino acid locations 64 and 74 was replaced with alanine) sensors. (D) RLuc signal measured from stable HEK293T cells expressing H3–K9 sensor transfected with scrambled and G9a specific SiRNAs. (E) RLuc signal measured in stable HEK293T cells expressing H3–K9 sensor transfected with scrambled and G9a specific siRNAs. (F) Immunoblot shows the level of dimethylated-H3–K9 sensor, endogenous dimethylated H3–K9, and G9a-methyltransferase measured in HEK293T cells transfected with SiRNA specific to G9a and scrambled-SiRNA. (G) Figure shows the change in the level of G9a-methyltransferase and dimethylated H3–K9 in HEK293T cells transfected with SiRNA specific to G9a-methyltransferase and scrambled-SiRNA.
Figure 4
Figure 4
Evaluation of histone methylation sensors (H3–K9 and H3–L9) in response to the treatment of different doses of methyltransferase and demethylase inhibitors in HEK293T cells stably expressing the sensors. (A) RLuc signal measured from stable HEK293T cells expressing H3–K9 sensor exposed to various concentrations (0 to 5.0 μM) of Bix01294. (B) RLuc signal measured from stable HEK293T cells expressing H3–L9 sensor exposed to various concentrations (0 to 5.0 μM) of Bix01294. (C) RLuc signal measured from stable HEK293T cells expressing H3–K9 sensor exposed to various concentrations (0 to 4.0 μM) of UNC0638. (D) RLuc signal measured from stable HEK293T cells expressing H3–L9 sensor exposed to various concentrations (0 to 4.0 μM) of UNC0638. (E) Immunoblot analysis of lysates of HEK293T cells stably expressing H3–K9 sensor treated with different doses of methyltransferase inhibitor (Bix01294) and demethylase inhibitor (JIB-04), for expressed H3–K9 sensor level, dimethylated fraction of H3–K9 sensor level, and endogenous dimethylated H3–K9 level. GAPDH was used to normalize the results. Dimethylated H3–K9 sensor protein was detected after immunoprecipitation of cell lysates using the tagged FLAG specific antibody. (F) RLuc sensor signal measured from HEK293T cells stably expressing H3–K9 sensor after treated with different doses of methyltransferase inhibitor (Bix01294) and demethylase inhibitor (JIB-04). Concentrations of Bix01294 and JIB-04 are labeled on the X-axis.
Figure 5
Figure 5
In vivo imaging of histone methylation in the nude mice model. (A) RLuc and FLuc signals of HEK293T xenograft expressing H3–K9 and H3–L9 sensors. (B) Normalized histone methylation assisted Renilla luciferase complementation signal measured from HEK293T xenograft expressing wild-type and mutant sensors. (C) RLuc and FLuc signals optically imaged from the tumor xenografts of HeLa cells stably expressing wild-type and mutant histone methylation sensors. (D) Normalized histone methylation assisted RLuc signal measured from HeLa xenograft expressing wild-type and mutant sensors.
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