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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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Characterization of COMET-mediated gene expression signatures in human cellsBG14 induces a light dose and compound concentration-dependent change in gene transcription in MCF-7 cells that is highly correlated with the gene expression signature of class I HDAC inhibitors. Specific examples of light-dependent up- and down-regulation of the HDAC inhibitor responsive landmark genes PAK1 (a) and CDK2 (b) in the presence of varying concentrations of BG14 and varying light exposure (no light, 250ms/750ms on/off, 750ms/250ms on/off (470 nm, 8.5 mW/cm2, 1 Hz) for 1 6 h. Data points represent values of 4 independent biological replicates. (c) Heat map of top 100 up- and down- regulated transcripts (landmark and inferred genes) that were specifically modulated by BG14 in light-dependent fashion. No meaningful transcriptional changes were observed by BG14 treatment alone or by light in the absence of inhibitor (control, 1 μM, 5 μM, 25μM; data represent average of n=4 measurements). (d) Gene Set Enrichment Analysis (GSEA) confirms the strongly correlated transcriptional response of CI-994 and BG14 (25 μM, 470 nm, 8.5 mW/cm2, 1 Hz, 75% duty cycle, 1 6 h). p < 0.001 and FDR q < 0.001 (e) Venn diagram showing the overlap between CI-994 and C60 response genes and BG14 regulated light-dependent “on-target” and light-independent “off-target” genes, respectively.
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Figure 5: Characterization of COMET-mediated gene expression signatures in human cellsBG14 induces a light dose and compound concentration-dependent change in gene transcription in MCF-7 cells that is highly correlated with the gene expression signature of class I HDAC inhibitors. Specific examples of light-dependent up- and down-regulation of the HDAC inhibitor responsive landmark genes PAK1 (a) and CDK2 (b) in the presence of varying concentrations of BG14 and varying light exposure (no light, 250ms/750ms on/off, 750ms/250ms on/off (470 nm, 8.5 mW/cm2, 1 Hz) for 1 6 h. Data points represent values of 4 independent biological replicates. (c) Heat map of top 100 up- and down- regulated transcripts (landmark and inferred genes) that were specifically modulated by BG14 in light-dependent fashion. No meaningful transcriptional changes were observed by BG14 treatment alone or by light in the absence of inhibitor (control, 1 μM, 5 μM, 25μM; data represent average of n=4 measurements). (d) Gene Set Enrichment Analysis (GSEA) confirms the strongly correlated transcriptional response of CI-994 and BG14 (25 μM, 470 nm, 8.5 mW/cm2, 1 Hz, 75% duty cycle, 1 6 h). p < 0.001 and FDR q < 0.001 (e) Venn diagram showing the overlap between CI-994 and C60 response genes and BG14 regulated light-dependent “on-target” and light-independent “off-target” genes, respectively.

Mentions: Having demonstrated light-dependent control of the acetylation state of the epigenome using COMET probes, we next sought to determine the biological consequence of HDAC inhibition at the level of the transcriptome in comparison to conventional light-independent HDAC inhibitors. To gain quantitative insights into the specific genes regulated by the COMET probes under a wide range of experimental conditions, including variation of compound dose, treatment time, and light exposure, we performed high-throughput gene expression profiling using the L1000 method from Genometry (Genometry, Inc.). The L1000 assay is a novel, high-throughput, multiplexed mRNA expression profiling technique developed as part of the NIH Common Fund supported LINCS project. It is based on the direct measurement of a reduced representation of the transcriptome (978 ‘landmark’ transcripts) and inference of the portion of the transcriptome not explicitly measured using an algorithm trained on tens of thousands of historical microarray-derived gene expression profiles. Eighty invariant transcripts are also explicitly measured to enable scaling and normalization. Specifically, we treated human MCF-7 cells and measured transcriptional changes as a function of compound concentration, light exposure, and treatment duration relative to the reference HDAC inhibitor CI-994. Light treatment alone or treatment with COMET probes in the absence of light had little to no effect on gene expression, while numerous light-dose and compound-concentration dependent transcriptional changes were observed in the presence of light. As expected for HDAC inhibition and consistent with previous reports, BG14 induced both up- and down-regulation of specific transcripts as illustrated by the examples of the directly measured landmark genes PAK1 and CDK2 (Fig. 5a, b).


Light Controlled Modulation of Gene Expression by Chemical Optoepigenetic Probes
Characterization of COMET-mediated gene expression signatures in human cellsBG14 induces a light dose and compound concentration-dependent change in gene transcription in MCF-7 cells that is highly correlated with the gene expression signature of class I HDAC inhibitors. Specific examples of light-dependent up- and down-regulation of the HDAC inhibitor responsive landmark genes PAK1 (a) and CDK2 (b) in the presence of varying concentrations of BG14 and varying light exposure (no light, 250ms/750ms on/off, 750ms/250ms on/off (470 nm, 8.5 mW/cm2, 1 Hz) for 1 6 h. Data points represent values of 4 independent biological replicates. (c) Heat map of top 100 up- and down- regulated transcripts (landmark and inferred genes) that were specifically modulated by BG14 in light-dependent fashion. No meaningful transcriptional changes were observed by BG14 treatment alone or by light in the absence of inhibitor (control, 1 μM, 5 μM, 25μM; data represent average of n=4 measurements). (d) Gene Set Enrichment Analysis (GSEA) confirms the strongly correlated transcriptional response of CI-994 and BG14 (25 μM, 470 nm, 8.5 mW/cm2, 1 Hz, 75% duty cycle, 1 6 h). p < 0.001 and FDR q < 0.001 (e) Venn diagram showing the overlap between CI-994 and C60 response genes and BG14 regulated light-dependent “on-target” and light-independent “off-target” genes, respectively.
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Figure 5: Characterization of COMET-mediated gene expression signatures in human cellsBG14 induces a light dose and compound concentration-dependent change in gene transcription in MCF-7 cells that is highly correlated with the gene expression signature of class I HDAC inhibitors. Specific examples of light-dependent up- and down-regulation of the HDAC inhibitor responsive landmark genes PAK1 (a) and CDK2 (b) in the presence of varying concentrations of BG14 and varying light exposure (no light, 250ms/750ms on/off, 750ms/250ms on/off (470 nm, 8.5 mW/cm2, 1 Hz) for 1 6 h. Data points represent values of 4 independent biological replicates. (c) Heat map of top 100 up- and down- regulated transcripts (landmark and inferred genes) that were specifically modulated by BG14 in light-dependent fashion. No meaningful transcriptional changes were observed by BG14 treatment alone or by light in the absence of inhibitor (control, 1 μM, 5 μM, 25μM; data represent average of n=4 measurements). (d) Gene Set Enrichment Analysis (GSEA) confirms the strongly correlated transcriptional response of CI-994 and BG14 (25 μM, 470 nm, 8.5 mW/cm2, 1 Hz, 75% duty cycle, 1 6 h). p < 0.001 and FDR q < 0.001 (e) Venn diagram showing the overlap between CI-994 and C60 response genes and BG14 regulated light-dependent “on-target” and light-independent “off-target” genes, respectively.
Mentions: Having demonstrated light-dependent control of the acetylation state of the epigenome using COMET probes, we next sought to determine the biological consequence of HDAC inhibition at the level of the transcriptome in comparison to conventional light-independent HDAC inhibitors. To gain quantitative insights into the specific genes regulated by the COMET probes under a wide range of experimental conditions, including variation of compound dose, treatment time, and light exposure, we performed high-throughput gene expression profiling using the L1000 method from Genometry (Genometry, Inc.). The L1000 assay is a novel, high-throughput, multiplexed mRNA expression profiling technique developed as part of the NIH Common Fund supported LINCS project. It is based on the direct measurement of a reduced representation of the transcriptome (978 ‘landmark’ transcripts) and inference of the portion of the transcriptome not explicitly measured using an algorithm trained on tens of thousands of historical microarray-derived gene expression profiles. Eighty invariant transcripts are also explicitly measured to enable scaling and normalization. Specifically, we treated human MCF-7 cells and measured transcriptional changes as a function of compound concentration, light exposure, and treatment duration relative to the reference HDAC inhibitor CI-994. Light treatment alone or treatment with COMET probes in the absence of light had little to no effect on gene expression, while numerous light-dose and compound-concentration dependent transcriptional changes were observed in the presence of light. As expected for HDAC inhibition and consistent with previous reports, BG14 induced both up- and down-regulation of specific transcripts as illustrated by the examples of the directly measured landmark genes PAK1 and CDK2 (Fig. 5a, b).

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