dCas9-mediated transcriptional activation of tissue inhibitor of metalloproteinases

Figures & data

Figure 1 Expression and transactivation of dCas9.nVP64.

Notes: (A) Cartoon illustration of the HA-tagged deactivated-form of dCas9.nVP64 was engineered at the N-terminus in the transactivating construct (dCas9.nVP64) while the non-transactivating construct was unlabeled (dCas9). Note that both dCas9 are HA-epitope tagged and the eGFP follows the 2A sequence resulting in cleavage of this reporter from the dCas9. (B) Western blot of overexpressed dCas9 from 3 replicate experiments. Anti-HA was used to detect dCas9 expression and anti-α-tubulin was used as a loading control. (C) Quantification of HA band intensity normalized by the α-tubulin loading control band intensity, mean±standard error of the mean, unpaired t-test, n=3. (D) Immunocytochemistry of cells expressing dCas9.nVP64 (top) or dCas9 (bottom). Phase images are on the left. The Hoechst nuclear stain (middle left, blue), the eGFP signal (middle, green), and the HA signal (middle right, red), are shown as a merged image on the right. Scale bar=50 mm. (E) Illustration of the reporter system used to detect transactivation. A 5xGAL4 promoter sequence is upstream of the mCherry reporter in the pG5.mCherry vector. As a control, GAL4-Id protein is expressed along with MyoD-VP16. GAL4 binds to the GAL4 promoter sequence and recruits VP16 upstream of the transcription start site (arrow) via the Id: MyoD interaction (CheckMate Two Hybrid, E2440; Promega, Madison, WI, USA). gRNA was engineered to target to the GAL4 promoter loci recruiting the dCas9.nVP64 upstream of the TSS driving expression of mCherry. (F) Fluorescence microscopy of cells expressing the pG5.mCherry promoter-reporter vector along with pGAL4.Id and pMyoD.VP16 (top), GAL4.gRNA and dCas9.nVP64 (middle), or GAL4.gRNA and dCas9 (bottom). Phase images are on the left. eGFP images indicate equal expression of both dCas9 and dCas9.nVP64 (middle left). mCherry signal, indicating the level of transactivation (middle right) while the merge analysis shows the dCas9.nVP64 transactivation is specific only to cells expressing the dCas9.nVP64 construct (right). Scale bar=100 mm.
Abbreviations: CMV, cytomegolovirus; eGFP, enhanced green fluorescent protein; HA, hemagglutinin; NLS, nuclear localization sequence; n.s., not significant; WPRE, Woodchuck hepatitis virus post-transcriptional regulatory element.

Figure 2 Loci-specific transactivation of the mouse TIMP promoters using dCas9.nVP64.

Notes: Fold-induction over the no gRNA transfection controls using dCas9.nVP64 (black circle) or dCas9 (orange triangle) and the relative gRNAs targeting loci within the (A) TIMP1, (B) TIMP2, or (C) TIMP3 core promoters. Closed symbols represent targeting of the top strand (also indicated by T) while open symbols represent targeting of the bottom strand (indicated by B) of the promoter DNA. X-axis shows the relative distance from the start codon and the numerous transcription factor-binding sites located within the mouse TIMP promoters. Data from Clark et al.²⁴ Mean±standard error of the mean, n=3.
Abbreviation: TIMP, tissue inhibitor of metalloproteinases.

Figure 3 Comparison of the transactivation efficiency of TIMP1 and TIMP2 promoters using different transcriptional activating effectors.

Notes: (A) Illustration of the different dCas9 constructs used. (B, C) Fold luciferase induction over no gRNA transfection controls comparing VP64-, VP160-, or VPR.dCas9 effector constructs targeting 3 loci within the mouse TIMP1 (B) or 4 loci within the mouse TIMP2 (C) promoter. Mean±standard error of the mean (SEM), *P<0.025, **P<0.005, ***P<0.0005, ****P<0.0001, unpaired t-test, n=4. (D, E) Fold luciferase induction over no gRNA transfection comparing VPR targeting 4 loci in the mouse TIMP1 (D) and mouse TIMP2 (E) promoter to SAM targeting between 0 and 250 bps upstream of the transcription start site. Mean±SEM, **P<0.01, ***P<0.005, one-way analysis of variance with multiple comparisons, n=4.
Abbreviations: CMV, cytomegolovirus; eGFP, enhanced green fluorescent protein; HA, hemagglutinin; NLS, nuclear localization sequence; SAM, synthetic activation motif; TIMP, tissue inhibitor of metalloproteinases; VPR, VP64-p65-Rta; WPRE, Woodchuck hepatitis virus post-transcriptional regulatory element.

Figure 4 Endogenous TIMP gene activation in mouse NSC34 cells.

Notes: (A) PMA stimulation enhances promoter-reporter activity for TIMP1 and 3. TIMP promoter-reporter constructs were transfected into HEK293 cells. The promoter activity (luciferase) was assessed by the Dual-Glo assay 12 hours after DMSO or PMA stimulation at the indicated concentrations. Mean±standard error of the mean (SEM), *P<0.05, **P<0.01 or n.s., n=3. (B) Endogenous TIMP mRNA basal expression in unstimulated NSC34 cells relative to GAPDH. Mean±SEM, n=6–11. All bars were significantly different (P<0,001) from each other by a pair-wise two-tailed t-test. Specific endogenous Cas9-VPR mediated transactivation using gRNAs targeting the mouse TIMP1 (C) and mouse TIMP2 (D) promoters. Dashed bar indicates the expression of all 4 gRNAs simultaneously. Mean±SEM, *P<0.05, **P<0.01, one-way analysis of variance with multiple comparisons, n=4. The “All” condition was analyzed separately; mean±SEM, ****P<0.0001, unpaired t-test, n=4.
Abbreviations: DMSO, dimethylsulfoxide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; n.s., not significant; PMA, phorbol-12-myristate-13-acetate; TIMP, tissue inhibitor of metalloproteinases; VPR, VP64-p65-Rta.

Figure 5 Production of functional TIMP2 protein by gRNA-induced NSC34 cells.

Notes: (A) Reverse zymography of cell culture supernatant. The lanes were loaded with control recombinant TIMP1 or 2 (10, 3,1, or 0 ng), or cell culture supernatant from NSC34 cells transfected with nothing, dCas-VPR alone, dCas-VPR with a negative control blank gRNA, or dCas-VPR with all 4 (p-194, p-535p, p-610, and p-956) TIMP2 gRNAs. The transfection condition was identical to that for mRNA assay except that the culture supernatant was harvested 48 hours after transfection. The blot is representative of 3 biological replicates. (B) Time course of increase in fluorescence due to cleavage of the fluorogenic MMP substrate. The slope of the fluorescence vs time plot was taken as a measure of the relative catalytic activity. The plot is representative of 3 biological replicate experiments. (C) A bar plot of relative MMP9 catalytic activity (mean±standard error of the mean, n=3). Note significant inhibition (****P<0.0001) of the catalytic activity by cell culture supernatant from wells transfected with all 4 TIMP2 gRNAs.
Abbreviations: MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinases; VPR, VP64-p65-Rta.

Clark IM, Swingler TE, Sampieri CL, Edwards DR. The regulation of matrix metalloproteinases and their inhibitors. Int J Biochem Cell Biol. 2008;40(6–7):1362–1378.

 PubMed Web of Science ®Google Scholar