Journal: Journal of Biological Chemistry

Article Title: Androglobin gene expression patterns and FOXJ1-dependent regulation indicate its functional association with ciliogenesis

doi: 10.1016/j.jbc.2021.100291

Figure Lengend Snippet: Figure 4. The upstream region of the ADGB transcriptional start site contains promoter activity and is inducible by CRISPRa. A, luciferase reporter assays of ADGB promoter (AP) elements of three different lengths from −33 bp to −2014 bp, −1064 bp, or −464 bp, respectively, upstream of the ADGB TSS in MCF-7 cells, HeLa cells, and HEK293T cells, showing consistent increase in ADGB promoter-driven luciferase activity in MCF-7 and HeLa (n = 3 inde- pendent experiments). Results are displayed as ratios of firefly to Renilla luciferase activities in relative light units (RLU) and normalized to results from pGL3- Basic control transfected cells. Schematic representation of cloned fragments upstream of the ADGB gene in a pGL3-Basic vector is shown with numbers representing positions corresponding to the first nucleotide of the TSS. B, HEK293T and MCF-7 cells were transfected with dCas9-VPR along with ADGB promoter (AP)-targeting gRNAs (gRNA AP-1 and/or gRNA AP-2) and ADGB promoter (pGL3B-AP464)-driven luciferase constructs. The gRNA used as negative control contains a nonspecific sequence as present in the pSPgRNA plasmid. Cas9-VPR-based activation of ADGB promoter (pGL3B-AP464)-driven luciferase constructs results in activation of the ADGB promoter construct (n = 3 independent experiments). Results are displayed as ratios of firefly to Renilla luciferase activities in RLU. C, HEK293T and MCF-7 cells were transfected with dCas9-VPR along with ADGB promoter-targeting gRNA AP-1 and/or gRNA AP-2, and relative ADGB transcript levels were quantified by RT-qPCR using a negative control gRNA as reference. Single-guide activation of the ADGB promoter with gRNA AP-1 and gRNA AP-2 results in substantial increment in ADGB transcript levels (n = 4 independent experiments). Simultaneous expression of gRNA AP- 1 and gRNA AP-2 leads to synergistic activation of endogenous ADGB expression (n = 4 independent experiments). D, immunoblotting of immunopre- cipitated ADGB from HEK293T and MCF-7 cells after gRNAs-dCas9-VPR-activation for 72 h detects endogenous ADGB expression. Data represent mean ± S.E.M (error bars); *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: All reporter gene assays were performed at least three times independently. dCas9-VPR-mediated activation of endogenous ADGB promoter Nuclease-null-Cas9 with tandem fusion of VP64-p65-Rta tripartite activator (dCas9-VPR, Addgene #63798) (24) was delivered along with gRNAs as described before (61) to activate ADGB promoter activity. gRNAs candidates targeting between −1 and −1700 bp upstream of ADGB TSS were cloned into, and expressed from, pSPgRNA plasmid (Addgene #47108), which was a generous gift from Prof. Charles Gersbach (62) (Table S2).

Techniques: Activity Assay, Luciferase, Control, Transfection, Clone Assay, Plasmid Preparation, Construct, Negative Control, Sequencing, Activation Assay, Quantitative RT-PCR, Expressing, Western Blot