Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Summary

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

Abstract

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation of the DNA-protein interaction, followed by immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-β (TGF-β)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

Introduction

In the nucleus of eukaryotes, DNA interacts with histone proteins and nonhistone proteins and is condensed into chromatin. In the physiological, pathophysiological, and cell biological context, cellular functions are spatially and temporarily controlled by chromatin-coordinated gene expression. DNA-protein interactions have an essential role in the regulation of cellular processes, such as DNA replication, recombination, and repair, as well as protein expression. Therefore, the analysis of DNA-protein interactions is an indispensable tool in the evaluation of gene expression and cell function.

Several techniques exist to assess DNA-protein ....

Protocol

1. Preparation of Solutions

1. Prepare the following solutions fresh for each experiment.
   1. For the fixation solution, prepare 37% formaldehyde and store it at RT. Dilute it in 1x Phosphate-Buffered Saline (PBS) to a final concentration of 1.42%.
      CAUTION: Formaldehyde is classified as irritant, corrosive, mutagenic, teratogenic, and carcinogenic. Do not ingest. Do not breathe gas/fumes/vapor/spray. In case of insufficient ventilation, wear suitable respiratory equipment. Avoid contact with skin and eyes. Keep away from incompatibles, such as oxidizing agents, reducing agents, acids, alkalis, and moisture.

Results

The binding of the TGF-β1 ligand to its cognate receptor complex results in the serine-phosphorylation of SMAD2/3 transcription factors, followed by their association with the common mediator, SMAD4. The SMAD complex translocates to the nucleus. TGF-β can regulate gene transcription, either directly via SMAD binding to SBEs within regulatory regions of the target genes, or indirectly through the SMAD-regulated expression of transcriptional activators or repressors that subsequently regula.......

Discussion

In this report, we demonstrate the TGF-β1-induced binding of SMAD2 to an SBE within the c-KIT ligand promoter and TGF-β1-induced binding of STAT3 to its recognition sequence within the TGF-β1 ligand gene. We demonstrate cytokine-induced binding of both transcription factors using chromatin immunoprecipitation.

Chromatin immunoprecipitation is a powerful tool to demonstrate the direct binding of a protein of interest to DNA, to characterize the stimuli that induce protein binding.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HepG2 cells	ATCC	HB-8065
Hep3B cells	ATCC	HB-8064
TGF-β1	R&D Systems	101-B1	Used at a concentration of 10 ng/mL
Anti-SMAD2 antibody	Cell Signalling Technology	5339	Amount used/IP: 3 µg
Anti-STAT3 antibody	Cell Signalling Technology	4904	Amount used/IP: 3 µg
ChIP-IT Protein G Magnetic Beads	Active Motif	53033
Protease Inhibitor Cocktail	Active Motif	37490
Micrococcal Nuclease	Cell Signalling Technology	10011
PCR forward primer: PAI-1			Sequence: 5’-GGAAGAGGATAAAGGACAAGCTG-3’
PCR reverse primer: PAI-1			Sequence: 5’-TGCAGCCAGCCACGTGATTGTC-3’
PCR forward primer: SCF			Sequence: 5’-CACTGATGTTAATGTTCAGC-3’
PCR reverse primer: SCF			Sequence: 5’-GCTCTAATTTAAACCTGGAGC-3’
PCR forward primer: TGF-β1 (STB-1)			Sequence: 5’-GAGAGAGACGTGAGTGGCATGTT-3’
PCR reverse primer: TGF-β1 (STB-1)			Sequence: 5’-TAGCTTTCTCTGCCTTGGTCTCCCC-3’
PCR forward primer: TGF-β1 (STB-2)			Sequence: 5’-GTACTGGGGGAGGAGCGGCATC-3’
PCR reverse primer: TGF-β1 (STB-2)			Sequence: 5’-TGCCACTGTCTGGAGAGAGGTGTGTC-3’