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Updated: Sep 28, 2026
Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets
Published on: July 7, 2010
[Identification of two new p53 target genes through implementation of the modified chromatin immunoprecipitation
R Burgess1, V Luniak, L Noskin
1TransGenetics Incorporated, University of California at San Diego, La Jolla, California, USA.
Abstract:
Direct target loci for the transcription factor p53 were identified through the employment of a combination of a modified version of chromosomal immunoprecipitation and inverse PCR. Irradiation of Hela cells to drive DNA damage response was followed by sequential chromosomal immunoprecipitation utilizing antibodies which recognize the large subunit of RNA polymerase II and p53. Inverse PCR with degenerate oligonucleotides specific for the p53 binding site was subsequently performed on immunoprecipitated DNA and fragments containing putative p53 target genes were subcloned and sequenced. Two sequences were identified which contain near-consensus p53 binding sites as well as recognition sites for the core transcriptional machinery including RNA polymerase II and Sp1. Cotransfections of vectors containing these sequences linked to a reporter with p53 expression vectors resulted in stimulation of transcription. Application of the technology described herein may result in the identification of target loci for a wide variety of transcription factors.
Insights
Researchers identified new p53 target genes using a novel ChIP-PCR method. This technique helps uncover direct targets for transcription factors, advancing our understanding of gene regulation.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Context:
- The transcription factor p53 plays a crucial role in DNA damage response and tumor suppression.
- Identifying direct p53 target genes is essential for understanding its regulatory functions.
Purpose:
- To develop and apply a novel method combining chromosomal immunoprecipitation (ChIP) and inverse PCR (iPCR) to identify direct p53 target loci.
- To validate the identified sequences as functional p53 targets through reporter assays.
Summary:
- A modified ChIP technique followed by iPCR was used on Hela cells after DNA damage induction.
- Antibodies against RNA polymerase II and p53 were employed for sequential immunoprecipitation.
- Two novel sequences with p53 binding sites and core transcriptional machinery recognition sites were identified and confirmed to stimulate transcription in reporter assays.
Impact:
- This study presents a powerful technology for discovering direct target genes of p53 and potentially other transcription factors.
- The findings contribute to a deeper understanding of the transcriptional regulatory networks controlled by p53.
- The developed methodology offers a valuable tool for future functional genomics and gene regulation studies.
