Gene Expression
Nebulas
Gene Expression NebulasSummary: While histone deacetylase (HDAC) inhibitors are thought to regulate gene expression by post-translational modification of histone as well as non-histone proteins. While histone hyperacetylation has long been considered the paradigmatic mechanism of action, recent genome-wide profiles indicate more complex interactions with the epigenome. In particular, HDAC inhibitors also induce histone deacetylation at the promoters of highly active genes, resulting in gene suppression. This was linked to the loss of histone acetyltransferase (HAT) binding. To illustrate pre-clinical utility of the HDAC inhibitor SAHA as a therapeutic, we show reversal of diabetes-associated EP300 target genes in diabetic HAECs of primary origin. These results were confirmed using SAHA, C646 (EP300/CREBBP inhibitor) or EP300 siRNA. These findings suggest the inhibition of gene expression by SAHA is mediated by EP300 function and provide a rationale for clinical trials of safety and efficacy in patients with diabetes.
Overall Design: Human aortic endothelial cells from a diabetic and non-diabetic individual were stimulated with DMSO (control), SAHA (2 μM, HDAC inhibitor) or C646 (10 μM, EP300 inhibitor) for 12 hours, or EP300 siRNA or non-target siRNA (control) for 4 hours, followed by 48 hours in fresh media. Study performed in triplicate.
| Strategy: |
|
| Species: |
|
| Tissue: |
|
| Healthy Condition: |
|
| Cell Type: |
|
| Development Stage: |
|
| Growth Protocol: | Human aortic endothelial cells (HAEC) from diabetic (CC-2919) and non-diabetic (CC-2535) individuals were maintained in endothelial cell basal growth media-2 (EBM-2, Lonza) with additional EBM-2 growth factors and supplements and with 10% heat-inactivated fetal bovine serum (Gibco). HAECs used in this study were between 5-9 passages. |
| Treatment Protocol: | Cells were stimulated with DMSO, SAHA (2 μM) or C646 (10 μM) for 12 hours. EP300 knock-down was generated using ON-TARGETplus Human EP300 (2033) siRNA – SMARTpool (L-003486-00-0005, Dharmacon). Cells were cultured in Opti-MEM® (Life Technologies) media with Lipofectamine® RNAiMAX Reagent (13778-075, Life Technologies) with either non-target (ON-TARGETplus Non-targeting Pool, D-001810-10-05, Dharmacon) or 25 nM EP300 siRNA for four hours. Media was replaced with EBM-2 and RNA was harvested after 48 hours. |
| Extract Protocol: | Total RNA was extracted in TRIzol (Invitrogen) and purified using the Direct-zolTM RNA MiniPrep kit (cat. no: R2052, Zymo Research) according to the manufacturers instructions.RNA was quantified on the MultiNA bioanalyzer (Shimadzu).NEBNext® Poly(A) mRNA Magnetic Isolation Module was used to enrich mRNA from 1 μg of total RNA. |
| Library Construction Protocol: | Libraries were quantified on the MultiNA Bioanalyzer (Shimadzu) and pooled to equimolar ratios for sequencing. |
| Molecule Type: | poly(A)+ RNA |
| Library Source: | |
| Library Layout: | SINGLE |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2500 |
| Strand-Specific: | Specific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
|---|