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.

SUBMITTER: Yang W 

PROVIDER: S-EPMC5504697 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature