Chromatin Immunoprecipitation Assay to Analyze the Effect of G-Quadruplex Interactive Agents on the Binding of RNA Polym
Growing evidence suggests the existence of G-quadruplexes and their involvement in transcriptional regulation of many human genes, including VEGF. These studies also provide strong evidence that G-quadruplex structures are stabilized by binding to small m
- PDF / 226,598 Bytes
- 10 Pages / 504.567 x 720 pts Page_size
- 19 Downloads / 165 Views
1
Introduction The chromatin immunoprecipitation (ChIP) technique is widely used to study protein-DNA interactions at the chromatin level in cultured cells [1]. This technique allows for the potential identification of proteins that associate with specific regions of the genome, including gene promoters [2]. The ChIP assay consists of a multistep process that is typically executed in a particular sequence [3]. Intact cells are first fixed with formaldehyde to covalently link protein to chromatin DNA, followed by mechanical shearing or enzymatic digestion of nucleoprotein complexes by sonication or Micrococcal Nuclease, respectively. The resulting cross-linked chromatin fragments are subjected to immunoprecipitation to enrich chromatin fragments where the target protein is present. The enriched chromatin complexes are then analyzed by
Danzhou Yang and Clement Lin (eds.), G-Quadruplex Nucleic Acids: Methods and Protocols, Methods in Molecular Biology, vol. 2035, https://doi.org/10.1007/978-1-4939-9666-7_13, © Springer Science+Business Media, LLC, part of Springer Nature 2019
233
234
Daekyu Sun
PCR amplification using gene-specific primers to detect and quantify specific DNA targets. Thus, the ChIP assay can be easily adopted as a powerful tool for characterizing mechanisms by which small molecules inhibit gene transcription, specifically by analyzing the binding of various transcription factors and RNA polymerase II (RNA Pol II) to gene promoter regions. Recently, G-quadruplex-interactive compounds have emerged as a new class of small molecules that inhibit the transcription of genes containing secondary DNA structures within their proximal promoter regions [4–6]. Since transcription factors and RNA Pol II are known to bind G-quadruplex-forming sequences in the proximal promoters of many genes in vivo [7], it is very important to determine how G-quadruplex interactive agents modulate transcription factor binding to these regions. The specific effect of Gquadruplex-interactive agents on gene transcription can be rationalized by demonstrating that these agents inhibit the recruitment of RNA Pol II and coregulators that assemble active transcription machinery. In recent studies, we have successfully utilized ChIP assays to determine how G-quadruplex-interactive agents affect RNA Pol II and transcription factor recruitment to target gene promoters [4–6]. We describe here the application of ChIP assays for examining the effect of G-quadruplex-interactive agents on the binding of RNA Pol II and other transcription factors to the VEGF promoter region, providing new insight into the role of these agents in VEGF transcriptional regulation. Analysis of the human VEGF promoter has revealed a proximal 36-bp region of a pPu/pPy tract (85 to 50), which was confirmed to form a stable intramolecular parallel G-quadruplex in vitro and in vivo in our previous studies [8, 9]. This region is functionally significant, as it contains three Sp1 transcription binding sites for mediating basal or inducible VEGF promoter activity [10
Data Loading...
