Citation

  • Authors: Rodriguez-Martinez A. et al.
  • Year: 2022
  • Journal: Biochim Biophys Acta Gene Regul Mech 1865 194811
  • Applications: in vitro / siRNA / INTERFERin
  • Cell types:
    1. Name: Hs 700T
      Description: Human pelvic adenocarcinoma cells
    2. Name: MCF-7

Method

siRNA transfections, qPCR and proliferation assay: ZNF414 siRNA was obtained from the Dharmacon siRNA library. Transfections were performed on 6-well plates for RNA-seq purposes and on 24-well plates for proliferation assays. Twenty-four hours after seeding, the cells were transfected with 10 nM siRNA using INTERFERin reagent (Polyplus Transfection) as per the manufacturer's instructions. An siRNA targeting the firefly luciferase (LUC) gene was used as a control in all experiments. Samples were collected 12 h and 24 h after transfection, and total RNA was extracted using a Nucleospin miRNA kit. qRT-PCR was performed as previously described [8]. The proliferation of MCF-7 cells was assessed using the AlamarBlue reagent as previously described

Abstract

Transcription factor binding to DNA is a central mechanism regulating gene expression. Thus, thorough characterization of this process is essential for understanding cellular biology in both health and disease. We combined data from three sequencing-based methods to unravel the DNA binding function of the novel ZNF414 protein in cells representing two tumor types. ChIP-exo served to map protein binding sites, ATAC-seq allowed identification of open chromatin, and RNA-seq examined the transcriptome. We show that ZNF414 is a DNA-binding protein that both induces and represses gene expression. This transcriptional response has an impact on cellular processes related to proliferation and other malignancy-associated functions, such as cell migration and DNA repair. Approximately 20% of the differentially expressed genes harbored ZNF414 binding sites in their promoters in accessible chromatin, likely representing direct targets of ZNF414. De novo motif discovery revealed several putative ZNF414 binding sequences, one of which was validated using EMSA. In conclusion, this study illustrates a highly efficient integrative approach for the characterization of the DNA binding and transcriptional activity of transcription factors.

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