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Light Controlled Modulation of Gene Expression by Chemical Optoepigenetic Probes

View Article: PubMed Central - PubMed

ABSTRACT

Epigenetic gene regulation is a dynamic process orchestrated by chromatin-modifying enzymes. Many of these master regulators exert their function through covalent modification of DNA and histone proteins. Aberrant epigenetic processes have been implicated in the pathophysiology of multiple human diseases. Small-molecule inhibitors have been essential to advancing our understanding of the underlying molecular mechanisms of epigenetic processes. However, the resolution offered by small molecules is often insufficient to manipulate epigenetic processes with high spatio-temporal control. Here, we present a novel and generalizable approach, referred to as ‘Chemo-Optical Modulation of Epigenetically-regulated Transcription’ (COMET), enabling high-resolution, optical control of epigenetic mechanisms based on photochromic inhibitors of human histone deacetylases using visible light. COMET probes may translate into novel therapeutic strategies for diseases where conditional and selective epigenome modulation is required.

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Optical properties and validation of light-dependent inhibitory activity toward recombinant HDACs of COMET probes(a) Measurement of thermal relaxation half-life. Each data point represents the mean values of 128 independent measurements. A least-squared fit modeling a one-phase decay (Graphpad Prism) was used to determine the relaxation half-lives. T1/2 values are reported with 95% Confidence Intervals. (b) 8x12 LED array for high-throughput COMET probe profiling. (c) Light intensity and concentration-dependent inhibition of recombinant HDAC3 deacetylase activity by BG14 (470 nm, Imax = 17 mW/cm2). Mean values of duplicate experiments. Dose dependent and light dependent (470 nm, 17 mW/cm2) activity of COMET probes and reference HDAC inhibitors against HDAC1(d), HDAC2(e) and HDAC3(f). Data represent mean values of duplicate experiments. Dose response estimation of HDAC target residence time of COMET probes. HDAC1 was incubated with BG47 (520 nM) (g) or BG48 (520 nM) (h) and exposed to blue light (470 nm, 17 mW/cm2) for 1 h and then kept for varying time in the dark at room temperature to allow HDAC bound inhibitor to dissociate. After the indicated time (4, 8, 17 h), HDAC substrate was added, and enzymatic activity was measured in kinetic mode over 2 h.(i) Enzymatic activity relative to control following light exposure and dark phase recovery. RU – relative units, RFU – relative fluorescence units. (g–i) Data represent individual measurements of representative biological replicate.
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Figure 2: Optical properties and validation of light-dependent inhibitory activity toward recombinant HDACs of COMET probes(a) Measurement of thermal relaxation half-life. Each data point represents the mean values of 128 independent measurements. A least-squared fit modeling a one-phase decay (Graphpad Prism) was used to determine the relaxation half-lives. T1/2 values are reported with 95% Confidence Intervals. (b) 8x12 LED array for high-throughput COMET probe profiling. (c) Light intensity and concentration-dependent inhibition of recombinant HDAC3 deacetylase activity by BG14 (470 nm, Imax = 17 mW/cm2). Mean values of duplicate experiments. Dose dependent and light dependent (470 nm, 17 mW/cm2) activity of COMET probes and reference HDAC inhibitors against HDAC1(d), HDAC2(e) and HDAC3(f). Data represent mean values of duplicate experiments. Dose response estimation of HDAC target residence time of COMET probes. HDAC1 was incubated with BG47 (520 nM) (g) or BG48 (520 nM) (h) and exposed to blue light (470 nm, 17 mW/cm2) for 1 h and then kept for varying time in the dark at room temperature to allow HDAC bound inhibitor to dissociate. After the indicated time (4, 8, 17 h), HDAC substrate was added, and enzymatic activity was measured in kinetic mode over 2 h.(i) Enzymatic activity relative to control following light exposure and dark phase recovery. RU – relative units, RFU – relative fluorescence units. (g–i) Data represent individual measurements of representative biological replicate.

Mentions: Thermal relaxation half-lives of push-pull azobenzene analogs similar to the presented compounds in aqueous solution at physiological pH have, to the best of our knowledge, not been reported. Previous approaches to study fast cis-to-trans isomerization are based on laser flash photolysis to measure the transient absorbance change following excitation with a short laser pulse24. Such experimental setups offer picosecond resolution; however, they are expensive and not commonly accessible. As an alternative, to measure relaxation kinetics with microsecond resolution, we designed a readily adaptable instrumentation setup (Supplementary Fig. 2 and Supplementary Fig. 3). Using this approach, we determined the thermal relaxation half-lives of HDAC ligands to range from 55–60 μs, relative to 47 μs for DABCYL, which is approximately 120× faster than previous measurements for DABCYL in chloroform (Fig. 2a) 17. The estimated distance of diffusion of small molecules (diffusion coefficient D = 10−6–10−7cm2/s) at 10×T1/2 (500 μs) in water is 0.1–0.3 μm, and is therefore expected to limit diffusion of “activated” inhibitors well within subcellular dimensions.


Light Controlled Modulation of Gene Expression by Chemical Optoepigenetic Probes
Optical properties and validation of light-dependent inhibitory activity toward recombinant HDACs of COMET probes(a) Measurement of thermal relaxation half-life. Each data point represents the mean values of 128 independent measurements. A least-squared fit modeling a one-phase decay (Graphpad Prism) was used to determine the relaxation half-lives. T1/2 values are reported with 95% Confidence Intervals. (b) 8x12 LED array for high-throughput COMET probe profiling. (c) Light intensity and concentration-dependent inhibition of recombinant HDAC3 deacetylase activity by BG14 (470 nm, Imax = 17 mW/cm2). Mean values of duplicate experiments. Dose dependent and light dependent (470 nm, 17 mW/cm2) activity of COMET probes and reference HDAC inhibitors against HDAC1(d), HDAC2(e) and HDAC3(f). Data represent mean values of duplicate experiments. Dose response estimation of HDAC target residence time of COMET probes. HDAC1 was incubated with BG47 (520 nM) (g) or BG48 (520 nM) (h) and exposed to blue light (470 nm, 17 mW/cm2) for 1 h and then kept for varying time in the dark at room temperature to allow HDAC bound inhibitor to dissociate. After the indicated time (4, 8, 17 h), HDAC substrate was added, and enzymatic activity was measured in kinetic mode over 2 h.(i) Enzymatic activity relative to control following light exposure and dark phase recovery. RU – relative units, RFU – relative fluorescence units. (g–i) Data represent individual measurements of representative biological replicate.
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Related In: Results  -  Collection

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Figure 2: Optical properties and validation of light-dependent inhibitory activity toward recombinant HDACs of COMET probes(a) Measurement of thermal relaxation half-life. Each data point represents the mean values of 128 independent measurements. A least-squared fit modeling a one-phase decay (Graphpad Prism) was used to determine the relaxation half-lives. T1/2 values are reported with 95% Confidence Intervals. (b) 8x12 LED array for high-throughput COMET probe profiling. (c) Light intensity and concentration-dependent inhibition of recombinant HDAC3 deacetylase activity by BG14 (470 nm, Imax = 17 mW/cm2). Mean values of duplicate experiments. Dose dependent and light dependent (470 nm, 17 mW/cm2) activity of COMET probes and reference HDAC inhibitors against HDAC1(d), HDAC2(e) and HDAC3(f). Data represent mean values of duplicate experiments. Dose response estimation of HDAC target residence time of COMET probes. HDAC1 was incubated with BG47 (520 nM) (g) or BG48 (520 nM) (h) and exposed to blue light (470 nm, 17 mW/cm2) for 1 h and then kept for varying time in the dark at room temperature to allow HDAC bound inhibitor to dissociate. After the indicated time (4, 8, 17 h), HDAC substrate was added, and enzymatic activity was measured in kinetic mode over 2 h.(i) Enzymatic activity relative to control following light exposure and dark phase recovery. RU – relative units, RFU – relative fluorescence units. (g–i) Data represent individual measurements of representative biological replicate.
Mentions: Thermal relaxation half-lives of push-pull azobenzene analogs similar to the presented compounds in aqueous solution at physiological pH have, to the best of our knowledge, not been reported. Previous approaches to study fast cis-to-trans isomerization are based on laser flash photolysis to measure the transient absorbance change following excitation with a short laser pulse24. Such experimental setups offer picosecond resolution; however, they are expensive and not commonly accessible. As an alternative, to measure relaxation kinetics with microsecond resolution, we designed a readily adaptable instrumentation setup (Supplementary Fig. 2 and Supplementary Fig. 3). Using this approach, we determined the thermal relaxation half-lives of HDAC ligands to range from 55–60 μs, relative to 47 μs for DABCYL, which is approximately 120× faster than previous measurements for DABCYL in chloroform (Fig. 2a) 17. The estimated distance of diffusion of small molecules (diffusion coefficient D = 10−6–10−7cm2/s) at 10×T1/2 (500 μs) in water is 0.1–0.3 μm, and is therefore expected to limit diffusion of “activated” inhibitors well within subcellular dimensions.

View Article: PubMed Central - PubMed

ABSTRACT

Epigenetic gene regulation is a dynamic process orchestrated by chromatin-modifying enzymes. Many of these master regulators exert their function through covalent modification of DNA and histone proteins. Aberrant epigenetic processes have been implicated in the pathophysiology of multiple human diseases. Small-molecule inhibitors have been essential to advancing our understanding of the underlying molecular mechanisms of epigenetic processes. However, the resolution offered by small molecules is often insufficient to manipulate epigenetic processes with high spatio-temporal control. Here, we present a novel and generalizable approach, referred to as ‘Chemo-Optical Modulation of Epigenetically-regulated Transcription’ (COMET), enabling high-resolution, optical control of epigenetic mechanisms based on photochromic inhibitors of human histone deacetylases using visible light. COMET probes may translate into novel therapeutic strategies for diseases where conditional and selective epigenome modulation is required.

No MeSH data available.


Related in: MedlinePlus