Figure 1.
(A) Schematic representation of the SREBP cleavage assay. (B) Activity of wild-type (WT) versus mutant (Mut) SRE promoter. HEK-293 cells were set up in a 96-well plate (in triplicate). After 24 hours, cells were transfected with either WT (open bars) or mutant (black bars) luciferase reporter constructs, along with renilla luciferase construct and the indicated plasmid /cDNA. Cells were grown for an additional 24 hours before performing the assay. (C) Effects of 25-hydroxy cholesterol (25-OH chol.) on SREBP signaling. The assay was carried out under varying 25-OH cholesterol concentrations (0.1–5 µg/ml) and for different incubation periods (6, 12, and 24 hours). 25-OH chol. was added to cells 1 day after transfecting with the reporter plasmids, SRE-luciferase and renilla luciferase. Maximum suppression of SRE-luciferase signals was observed after 24 hours of incubation with 25-OH chol (shown here). The effects of DP-SCAP under high 25-OH cholesterol levels are significantly higher at all concentrations (Student's t-Test, p<0.005). (D) Effects of known repressors, activators and high cholesterol (25OH chol., 1 µg/ml) on the SREBP signaling pathway. The assay was carried out as in B. Basal refers to pcDNA3 overexpression. SCAP and DP-SCAP significantly activate the assay in the absence (white bars) or presence (black bars) of 25-OH cholesterol (Student's t-Test, p<0.05). (B–D) Error bars indicate standard deviations (n = 3). Where not visible, error bars are smaller than symbols. The graphs are representative of at least 2 independent experiments.
Figure 2.
Primary and secondary screen results.
(A) Scatter plot showing the result from the primary screening of 10,000 putative full-length human cDNA's in the SREBP cleavage assay. The 2D-normalized z-scores for a clone in the first experiment (x-axis) are plotted against that obtained in the second experiment (y-axis). Genes at the top right corner represent potential activators of SREBP signaling, while those at the bottom left corner represent potential repressors of SREBP signaling (circles). (B) Scatter plot representing the combined data from all secondary screens. Each clone was re-tested in triplicate in two separate experiments. Firefly to renilla luciferase ratios for a clone in the first experiment (x-axis) were plotted against the ratios for the same clone in the second experiment (y-axis). (C) Analysis of the selected 176 clones under conditions identical to those used in the primary screen. Scatter plot shows luciferase ratios obtained for a clone in the first experiment against ratios obtained for the same clone in the second experiment. Activators are represented in red and repressors in blue. Circles represent clones displaying the highest activation or repression of luciferase ratios. (D) Effect of the 176 selected activators and suppressors (grey points) on mutant SRE promoter. The scatter plot shows the luciferase ratios obtained for a clone in the first experiment against luciferase ratios obtained in the second experiment. Central data points (circle) represent genes that did not have an effect on the mutant SRE-luciferase. Grey points falling at the extremities represent clones that activated the mutant SRE-luciferase or had higher levels of renilla luciferase. Control genes are color coded as: red, DN-SCAP; dark blue, DP-SCAP; yellow, INSIG1; black, SCAP; green, pSport6; sky blue, pXL4; pink, pcDNA3.
Figure 3.
Distribution of 2D normalized z-scores (NZ2D) for (A) all cDNA clones used in this screen and clones assigned to the (B) N-3,6 Polyunsaturated fatty acid biosynthesis, (C) Cell Adhesion / Cell Matrix Interaction and (D) Gap Junction pathways. The rightward shift of NZ2D scores among fatty acid synthesis genes (B) relative to background (A) indicates that overexpression of genes in this pathway on balance tend to activate SREBP transcriptional activity, whereas the leftward shift in (C) indicates that the cell adhesion/cell matrix tend to inhibit SREBP transcriptional activity in this screen. Gap junction genes were spread on either side of the median (D) indicating that a sub-set of gap junction genes activated the SREBP pathway, while others repressed it.
Table 1.
List of pathways designated as activators of SREBP signaling by Wilcoxon test.
Table 2.
List of pathways designated as repressors of SREBP signaling by Wilcoxon test.
Table 3.
List of SREBP pathway modulators by LBF test.
Figure 4.
Novel activators of the SREBP pathway.
(A) Effects of activator genes in presence of 25-OH cholesterol. The cDNA's were assayed in the presence of 1 µg/ml 25-hydroxy cholesterol. Scatter plot shows the luciferase ratios obtained for a clone in the first experiment against that obtained in the second experiment. Data points lying at the bottom left corner of the scatter plot represent genes that do not activate SRE-luciferase in the presence of high cholesterol. SCAP (black points), DP-SCAP (dark blue points) and SREBF1 overcome cholesterol repression (top right corner). Empty vectors color coded as: green, pSport6; sky blue, pXL4; pink, pcDNA3. (B) Effects of INSIG1 overexpression (15 ng/well) on novel activator genes. SCAP (central black points), DP-SCAP (dark blue points) and SREBF1 (top right corner) escape repression by INSIG1. In addition, a number of candidate genes (grey points) activated SRE-luciferase to a greater extent than SCAP. Colored points represent empty vectors: green, pSport6; sky blue, pXL4; pink, pcDNA3. (C) Graphical representation of the ten novel activator genes that activate SREBP signaling under high INSIG1 levels. These genes activate the SREBP signaling assay to a significantly greater extent than SCAP (Student's t-Test, p<0.005 or p<0.05).
Figure 5.
Novel repressors of the SREBP pathway.
(A) Effects of SCAP (15 ng/well) over-expression on novel repressor genes. Scatter plot depicting the luciferase ratios obtained for a clone in the first experiment against ratios obtained in the second experiment. Internal control genes are color coded as: yellow, INSIG1; red, DN-SCAP; green, pSport6; sky blue, pXL4; pink, pcDNA3. (B) Graphical representation of eight repressors that can inhibit SCAP-mediated SREBP activation as efficiently as INSIG1 (Student's t-Test, p>0.01).
Table 4.
Overlap between GSEA and biological validation.