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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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Light-dependent control of the human epigenome with COMET probes(a) Immunofluorescence analysis of H3K9ac levels in MCF-7 cells treated with CI-994 (25 μM) and BG14 (25 μM) in presence and absence of light (470 nm, 8.5 mW/cm2, 1Hz, 75% duty cycle). Scale bar = 20μm. (b) Quantification of H3K9ac intensity changes by automated confocal microscopy. Mean values +s.d. are shown (n=3). Significance values (n.s. – not significant, *** p < 0.001) were calculated by one-way ANOVA with Tukey’s post-hoc test (Graphpad PRISM). (c–e) Western blot analysis of H3K9ac levels in MCF-7 cells after treatment with COMET probes at 25 μM and varying amounts of light exposure (Supplementary Fig. 8). Specifically, cells were exposed to no light or pulsing light (470 nm, 8.5 mW/cm2) modulated at a frequency of 1 Hz (the duty cycle is expressed in % ON time). Values were normalized to vehicle treated cells and represent duplicate means of biological duplicates. (f) Persistence of HDAC inhibition. Quantification of relative immunofluorescence intensity of H3K9ac upon treatment of MCF-7 cells with DMSO, CI-994 (25 μM) and BG14 (25 μM) for a total of 16 h experimental time under varying duration of light exposure (470 nm, 8.5 mW/cm2, 1Hz, 50% duty cycle) at the beginning of the treatment period, followed by varying length of dark phases. Cellular H3K9 acetylation levels were normalized within each group to the positive control CI-994 = 100% and DMSO = 0. Mean values +s.d. are shown (n≥3). Detailed statistical values are provided in Supplementary Figure 8.
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Figure 3: Light-dependent control of the human epigenome with COMET probes(a) Immunofluorescence analysis of H3K9ac levels in MCF-7 cells treated with CI-994 (25 μM) and BG14 (25 μM) in presence and absence of light (470 nm, 8.5 mW/cm2, 1Hz, 75% duty cycle). Scale bar = 20μm. (b) Quantification of H3K9ac intensity changes by automated confocal microscopy. Mean values +s.d. are shown (n=3). Significance values (n.s. – not significant, *** p < 0.001) were calculated by one-way ANOVA with Tukey’s post-hoc test (Graphpad PRISM). (c–e) Western blot analysis of H3K9ac levels in MCF-7 cells after treatment with COMET probes at 25 μM and varying amounts of light exposure (Supplementary Fig. 8). Specifically, cells were exposed to no light or pulsing light (470 nm, 8.5 mW/cm2) modulated at a frequency of 1 Hz (the duty cycle is expressed in % ON time). Values were normalized to vehicle treated cells and represent duplicate means of biological duplicates. (f) Persistence of HDAC inhibition. Quantification of relative immunofluorescence intensity of H3K9ac upon treatment of MCF-7 cells with DMSO, CI-994 (25 μM) and BG14 (25 μM) for a total of 16 h experimental time under varying duration of light exposure (470 nm, 8.5 mW/cm2, 1Hz, 50% duty cycle) at the beginning of the treatment period, followed by varying length of dark phases. Cellular H3K9 acetylation levels were normalized within each group to the positive control CI-994 = 100% and DMSO = 0. Mean values +s.d. are shown (n≥3). Detailed statistical values are provided in Supplementary Figure 8.

Mentions: Following the biochemical characterization of the COMET probes, we profiled BG14, BG47, and BG48 in cellular assays using acetylation of histone H3 Lysine 9 (H3K9ac) as a pharmacodynamic marker for HDAC inhibition. As shown in Figure 3, treatment of MCF-7 cells (a human breast cancer cell line) with BG14 in the presence of light for 16 hours strongly induced acetylation of H3K9 relative to the vehicle control comparable to CI-994 treatment in an immunofluorescence assay (Fig. 3 a, b). H3K9 acetylation levels directly correlated with total light exposure (Fig. 3 c–e, Supplementary Fig. 8) as quantified by western blot analysis. Independently of the assay method, we did not observe significant increase in histone acetylation in the absence of light. As expected, BG14, which also inhibited HDAC3, induced the strongest increase of H3K9ac (Fig. 3c) compared to the HDAC1/2-selective inhibitors BG47 (Fig. 3d) and BG48 (Fig. 3e). These results with COMET probes demonstrated, for the first time, light-dependent control of the human epigenome.


Light Controlled Modulation of Gene Expression by Chemical Optoepigenetic Probes
Light-dependent control of the human epigenome with COMET probes(a) Immunofluorescence analysis of H3K9ac levels in MCF-7 cells treated with CI-994 (25 μM) and BG14 (25 μM) in presence and absence of light (470 nm, 8.5 mW/cm2, 1Hz, 75% duty cycle). Scale bar = 20μm. (b) Quantification of H3K9ac intensity changes by automated confocal microscopy. Mean values +s.d. are shown (n=3). Significance values (n.s. – not significant, *** p < 0.001) were calculated by one-way ANOVA with Tukey’s post-hoc test (Graphpad PRISM). (c–e) Western blot analysis of H3K9ac levels in MCF-7 cells after treatment with COMET probes at 25 μM and varying amounts of light exposure (Supplementary Fig. 8). Specifically, cells were exposed to no light or pulsing light (470 nm, 8.5 mW/cm2) modulated at a frequency of 1 Hz (the duty cycle is expressed in % ON time). Values were normalized to vehicle treated cells and represent duplicate means of biological duplicates. (f) Persistence of HDAC inhibition. Quantification of relative immunofluorescence intensity of H3K9ac upon treatment of MCF-7 cells with DMSO, CI-994 (25 μM) and BG14 (25 μM) for a total of 16 h experimental time under varying duration of light exposure (470 nm, 8.5 mW/cm2, 1Hz, 50% duty cycle) at the beginning of the treatment period, followed by varying length of dark phases. Cellular H3K9 acetylation levels were normalized within each group to the positive control CI-994 = 100% and DMSO = 0. Mean values +s.d. are shown (n≥3). Detailed statistical values are provided in Supplementary Figure 8.
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Figure 3: Light-dependent control of the human epigenome with COMET probes(a) Immunofluorescence analysis of H3K9ac levels in MCF-7 cells treated with CI-994 (25 μM) and BG14 (25 μM) in presence and absence of light (470 nm, 8.5 mW/cm2, 1Hz, 75% duty cycle). Scale bar = 20μm. (b) Quantification of H3K9ac intensity changes by automated confocal microscopy. Mean values +s.d. are shown (n=3). Significance values (n.s. – not significant, *** p < 0.001) were calculated by one-way ANOVA with Tukey’s post-hoc test (Graphpad PRISM). (c–e) Western blot analysis of H3K9ac levels in MCF-7 cells after treatment with COMET probes at 25 μM and varying amounts of light exposure (Supplementary Fig. 8). Specifically, cells were exposed to no light or pulsing light (470 nm, 8.5 mW/cm2) modulated at a frequency of 1 Hz (the duty cycle is expressed in % ON time). Values were normalized to vehicle treated cells and represent duplicate means of biological duplicates. (f) Persistence of HDAC inhibition. Quantification of relative immunofluorescence intensity of H3K9ac upon treatment of MCF-7 cells with DMSO, CI-994 (25 μM) and BG14 (25 μM) for a total of 16 h experimental time under varying duration of light exposure (470 nm, 8.5 mW/cm2, 1Hz, 50% duty cycle) at the beginning of the treatment period, followed by varying length of dark phases. Cellular H3K9 acetylation levels were normalized within each group to the positive control CI-994 = 100% and DMSO = 0. Mean values +s.d. are shown (n≥3). Detailed statistical values are provided in Supplementary Figure 8.
Mentions: Following the biochemical characterization of the COMET probes, we profiled BG14, BG47, and BG48 in cellular assays using acetylation of histone H3 Lysine 9 (H3K9ac) as a pharmacodynamic marker for HDAC inhibition. As shown in Figure 3, treatment of MCF-7 cells (a human breast cancer cell line) with BG14 in the presence of light for 16 hours strongly induced acetylation of H3K9 relative to the vehicle control comparable to CI-994 treatment in an immunofluorescence assay (Fig. 3 a, b). H3K9 acetylation levels directly correlated with total light exposure (Fig. 3 c–e, Supplementary Fig. 8) as quantified by western blot analysis. Independently of the assay method, we did not observe significant increase in histone acetylation in the absence of light. As expected, BG14, which also inhibited HDAC3, induced the strongest increase of H3K9ac (Fig. 3c) compared to the HDAC1/2-selective inhibitors BG47 (Fig. 3d) and BG48 (Fig. 3e). These results with COMET probes demonstrated, for the first time, light-dependent control of the human epigenome.

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 &lsquo;Chemo-Optical Modulation of Epigenetically-regulated Transcription&rsquo; (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