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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.

No MeSH data available.


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Light-dependent spatial control of epigenetic regulation with the COMET probe BG14Quantification of relative immunofluorescence intensity of H3K9ac upon treatment of MCF-7 cells with BG14 and light. (a) MCF-7 cells in a 1-well chamber slide were placed over a 5 mm LED (470 nm, 8.5 mW/cm2, 0.9 Hz, 33% duty cycle for 6 h, followed by 10 h without light exposure). The heatmap represents relative levels of histone H3K9ac (red indicates elevated H3K9ac levels). The white circle indicates position of LED. (b) Wells of a 96-well culture plate were partially covered at the bottom of the plate prior to treatment of MCF-7 cells with DMSO control, BG14 and CI-994 in presence of light for 16 h followed by immunofluorescence staining for H3K9ac. Heatmaps of H3K9ac intensity throughout wells. Scale bar = 1mm. (c) Quantification of H3K9ac spatial intensity differences by automated, confocal microscopy. Mean values +s.d. (n=5). Significance values (n.s. – not significant, * p < 0.05, *** p < 0.001) were calculated by one-way ANOVA with Tukey’s post-hoc test (Graphpad PRISM).
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Figure 4: Light-dependent spatial control of epigenetic regulation with the COMET probe BG14Quantification of relative immunofluorescence intensity of H3K9ac upon treatment of MCF-7 cells with BG14 and light. (a) MCF-7 cells in a 1-well chamber slide were placed over a 5 mm LED (470 nm, 8.5 mW/cm2, 0.9 Hz, 33% duty cycle for 6 h, followed by 10 h without light exposure). The heatmap represents relative levels of histone H3K9ac (red indicates elevated H3K9ac levels). The white circle indicates position of LED. (b) Wells of a 96-well culture plate were partially covered at the bottom of the plate prior to treatment of MCF-7 cells with DMSO control, BG14 and CI-994 in presence of light for 16 h followed by immunofluorescence staining for H3K9ac. Heatmaps of H3K9ac intensity throughout wells. Scale bar = 1mm. (c) Quantification of H3K9ac spatial intensity differences by automated, confocal microscopy. Mean values +s.d. (n=5). Significance values (n.s. – not significant, * p < 0.05, *** p < 0.001) were calculated by one-way ANOVA with Tukey’s post-hoc test (Graphpad PRISM).

Mentions: To demonstrate spatial control within a cell population, we exposed MCF-7 cells grown in a 1-well chamber slide in the presence of BG14. Using our 96-well LED array, we exposed one side to blue light from a single LED for 6 h followed by 10 h in the dark and quantified histone H3K9ac by immunofluorescence staining. As shown in Figure 4a, significant increase of H3K9 acetylation was limited to the light exposed areas within the same chamber.


Light Controlled Modulation of Gene Expression by Chemical Optoepigenetic Probes
Light-dependent spatial control of epigenetic regulation with the COMET probe BG14Quantification of relative immunofluorescence intensity of H3K9ac upon treatment of MCF-7 cells with BG14 and light. (a) MCF-7 cells in a 1-well chamber slide were placed over a 5 mm LED (470 nm, 8.5 mW/cm2, 0.9 Hz, 33% duty cycle for 6 h, followed by 10 h without light exposure). The heatmap represents relative levels of histone H3K9ac (red indicates elevated H3K9ac levels). The white circle indicates position of LED. (b) Wells of a 96-well culture plate were partially covered at the bottom of the plate prior to treatment of MCF-7 cells with DMSO control, BG14 and CI-994 in presence of light for 16 h followed by immunofluorescence staining for H3K9ac. Heatmaps of H3K9ac intensity throughout wells. Scale bar = 1mm. (c) Quantification of H3K9ac spatial intensity differences by automated, confocal microscopy. Mean values +s.d. (n=5). Significance values (n.s. – not significant, * p < 0.05, *** p < 0.001) were calculated by one-way ANOVA with Tukey’s post-hoc test (Graphpad PRISM).
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Figure 4: Light-dependent spatial control of epigenetic regulation with the COMET probe BG14Quantification of relative immunofluorescence intensity of H3K9ac upon treatment of MCF-7 cells with BG14 and light. (a) MCF-7 cells in a 1-well chamber slide were placed over a 5 mm LED (470 nm, 8.5 mW/cm2, 0.9 Hz, 33% duty cycle for 6 h, followed by 10 h without light exposure). The heatmap represents relative levels of histone H3K9ac (red indicates elevated H3K9ac levels). The white circle indicates position of LED. (b) Wells of a 96-well culture plate were partially covered at the bottom of the plate prior to treatment of MCF-7 cells with DMSO control, BG14 and CI-994 in presence of light for 16 h followed by immunofluorescence staining for H3K9ac. Heatmaps of H3K9ac intensity throughout wells. Scale bar = 1mm. (c) Quantification of H3K9ac spatial intensity differences by automated, confocal microscopy. Mean values +s.d. (n=5). Significance values (n.s. – not significant, * p < 0.05, *** p < 0.001) were calculated by one-way ANOVA with Tukey’s post-hoc test (Graphpad PRISM).
Mentions: To demonstrate spatial control within a cell population, we exposed MCF-7 cells grown in a 1-well chamber slide in the presence of BG14. Using our 96-well LED array, we exposed one side to blue light from a single LED for 6 h followed by 10 h in the dark and quantified histone H3K9ac by immunofluorescence staining. As shown in Figure 4a, significant increase of H3K9 acetylation was limited to the light exposed areas within the same chamber.

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