A novel strategy to dissect endogenous gene transcriptional regulation in live cells

Abstract

Gene transcription is a central tenet of biology, traditionally measured by RT-PCR, microarray, or more recently, RNA sequencing. However, these measurements only provide a snapshot of the state of gene transcription and only represent an overall readout of complex transcriptional networks that regulate gene expression. In this report, we describe a novel strategy to dissect endogenous gene transcription regulation in live cells by knocking in a reporter gene, EGFP, under the control of the endogenous gene promoter, using the ARID1A gene as an example. The ARID1A gene, encoding a subunit of the ATP-dependent chromatin remodeling complex SNF/SWI, has recently been identified as a tumor suppressor in multiple cancers. Despite studies that elucidate the mechanism of ARID1A's tumor suppressor function, little is known of the genes/events that regulate ARID1A expression. Using the HEK293 cells as a model, we discovered novel aspects of ARID1A transcription regulation in response to cell cycle progression, DNA damage, and microRNAs, exemplifying the potential of our strategy in providing new insight to the mechanism of gene transcription regulation. This strategy can be generalized to essentially any gene of interest, making it a powerful tool for the study of gene expression heterogeneity, especially in cancer cells, and a robust readout for high-throughput screening of agents that modulate gene transcription.

Keywords: ARID1A; CRISPR-Cas9; Transcription regulation.

Figure 1. Establishing a model that monitors endogenous ARID1A gene transcription

A. Schematic view of our knockin strategy. In our study, the ORF of EGFP (717 bp) was introduced into the ARID1A exon 2 in frame. The ATG start codon of both ORFs is truncated. There are two alternative splicing products of ARID1A. The ATG start codon labeled in black is for the longer transcript, while the one in blue is for the shorter transcript. The knockin was purposely placed in the middle of the ATG start codon for the shorter transcript to destroy it, as indicated by a cross, to ensure that the reporter gene does not interfere with normal ARID1A gene splicing and to keep the regulation of ARID1A transcription by the endogenous promoter intact. The CRISPR-Cas9 target site is indicated by a red asterisk on exon 2. The red bars flanking the reporter ORF in the donor plasmid represent the 1-kb 5′ and 3′ homologous arms to ARID1A genomic locus. B. EGFP-positive clones without sorting (top panel). Fluorescence imaging demonstrates that the number of false EGFP-positive clones is high when cells are cultured for 1 week following the first round of sorting (middle panel). The red circles highlight false-positive cells. However, every cell among the clones, following the second round of sorting, is EGFP positive (lower panel). Scale bar, 200 μm. C. DNA sequencing chromatograms indicate successful knockin of the EGFP gene at the expected integration site. The stop codon of the EGFP gene is underlined by a red bar.

Figure 2. Cell cycle regulation of ARID1A transcription

A. Successful synchronization of cell cycle by serum deprivation, aphidicolin, and nocodazole as indicated by flow cytometry analysis. Unsynchronized cells were used as a control. B. Regulation of ARID1A expression, as indicated by EGFP intensity, is cell cycle–dependent. Cells are synchronized by serum deprivation at G0 phase, aphidicolin at S phase, and nocodazole at M phase. M phase has the strongest EGFP signal, while G0 has the least. The bar in the synchronized cell plot indicates a drastic difference of the EGFP signal between G0 and M phases, which translates into dramatic change of ARID1A transcription between these two phases of cell cycle. C. RT-qPCR results demonstrate changes of ARID1A levels at different phases of cell cycle. Error bar, standard deviation (sd) from three independent RT-qPCR experiments. *** p<0.0005. D. Fixed fluorescent and live cell imaging of EGFP expression in cells synchronized at different phases of the cell cycle. It is clear that G2/M phase has the strongest EGFP signal, while G0 has the weakest. Interestingly, the EGFP signal is located inside the nucleus, suggesting a cryptic nuclear localization signal in the first 393 aa of the ARID1A protein. Scale bar, 50 μm for the top two panels; 20 μm for the lower panel. At least five randomly selected view fields were imaged and the representative images were shown in the panel.

Figure 3. Regulation of ARID1A transcription by DNA damage signal

A. CPT induced robust DNA damage, demonstrated by phosphorylation of serine 15 (s-15) of TP53 (p-p53) and induction of p21 by Western blotting. Endogenous ARID1A produced from the wild type allele is inhibited by DNA damage signals, which are also faithfully reflected by downregulation of EGFP reporter. B. Flow cytometry analysis of EGFP expression in HEK293 cells treated by 6 μM CPT at 0, 1, 4, and 8 hrs. A clear suppression of fluorescence signal can be detected after 4 hours of CPT treatment. C. Fluorescence imaging in live cells treated by 6 μM CPT at 0, 1, 4, and 8 hrs demonstrates the suppression of EGFP expression. Interestingly, loss of EGFP is seen in nucleoli after 8 hours of CPT treatment. Scale bar, 20 μm. At least five randomly selected view fields were imaged and the representative images were shown in the panel. D. RT-qPCR results demonstrate inhibition of endogenous ARID1A and reporter EGFP transcription following DNA damage at different time points (hrs). Error bar, standard deviation (sd) from three independent RT-qPCR experiments. ** p<0.01; *** p<0.0005.

Figure 4. Regulation of ARID1A transcription by miR-144 and miR-9

A. Fluorescence imaging in live cells demonstrates the suppression of EGFP expression following transfection of miRNA mimics. A scramble mimic was also used as a control. Scale bar, 50 μm. At least five randomly selected view fields were imaged and the representative images were shown in the panel. B. Flow cytometry analysis confirms the decrease of fluorescence intensity from the cells treated with miRNA mimics. C. Both endogenous ARID1A and reporter EGFP proteins are downregulated by miRNA mimics. D. RT-qPCR results demonstrate inhibition of endogenous ARID1A and reporter EGFP transcription by miRNA mimics, suggesting that miR-9 and miR-144 regulate ARID1A primarily through mRNA degradation. Error bar, standard deviation (sd) from three independent RT-qPCR experiments. * p<0.05.