Chromatin Immunoprecipitation (ChIP)

What is ChIP?

A very informative technique that allows analysis of protein function and epigenetic changes during the process of transcription. Can also be applied to study other cellular events that requires chromatin processing, such as DNA replication and repair.

What does ChIP rely on?

The enrichment of a target protein using an antibody in a process called immunoprecipitation.

What is transcription?

The process in which single-stranded RNA is generated from complementary DNA sequence within the nucleus of the cell.

What is RNA polymerase II?

The enzyme that transcribes protein-coding genes within DNA into RNA

What is RNA polymerase I?

Involved in transcribing ribosomal RNAs

What is RNA polymerase III?

Involved in transcribing tRNAs

What are transcription factors?

Facilitate the loading of RNA polymerase onto target gene promoter elements allowing for transcription to take place.

Why are proteins required for transcription?

DNA is largely inaccessible to cellular processing due to the extent of packaging required to fit it into the nucleus. Proteins capable of facilitating access of RNA polymerase to DNA are recruited to promoter elements.

What is the nucleosome?

Basic unit of chromatin that contains 147bp of DNA wrapped around a core of proteins called the histone octamer?

What is the histone octamer?

2 copies of histones H2A, H2B, H3 and H4 make up this core protein complex. Compacted nucleosomal DNA is transcriptionally inert. For transcription to take place, remodelling of nucleosomes must occur to permit access for RNA polymerase binding.

What are the transcriptional effects?

Histone acetylation activates. Histone methylation on histone H3-Lysine 4 activates. Histone methylation on histone H3-Lysine 9 represses. Histone acetyltransferases catalyse histone acetylation. Histone methyltransferases catalyse histone methylation

What is the ChIP Procedure?

Cross-link protein and DNA with formaldehyde.

Lyse cells and sonicate to fragment chromatin.

Add antibody-bound beads to bind protein-DNA complexes (immunoprecipitation).

Reverse cross-links between protein-DNA and Proteinase K treat samples to remove protein from mixture.

Purify DNA and set up quantitative PCR or DNA sequencing.

What is step 1 of ChIP?

Cross-linking protein-DNA and protein-protein interactions

What happens in step 1?

Formaldehyde introduces covalent cross-links between DNA and protein that sticks them together. For transcription factors, cross-linking them to DNA is very important as their interaction with promoter elements is ultimately transient and may not be detected otherwise.

What is step 2 of ChIP?

Cell lysis and chromatin fragmentation by sonication

What happens in step 2?

Formaldehyde-treated cells are lysed using a series of buffers to enrich for nuclear proteins. Samples are then sonicated- a process in which high-frequency sonic waves are directed onto each sample to break phosphodiester bonds within DNA. This process results in fragmented chromatin that retain the cross-linked DNA-protein interactions introduced in step 1. The more sonication of each sample, the smaller the chromatin fragments: ideal size is between 200-500 base-pairs of DNA

what is step 3 of ChIP?

Immunoprecipitation

What happens in step 3?

Antibodies targeted to a specific protein or a control IgG are mixed with magnetic beads and subsequently added to the mixture. Antibody binds to the target protein complexed to DNA and enriches the protein-DNA complex using magnetic-based separation

What are steps 4 and 5?

Cross-link reversal and proteinase K treatment

What happens in step 4 & 5?

After immunoprecipitation, the antibody-DNA/protein complex is washed and then treated with salt buffer and heated to reverse the covalent cross-links between protein and DNA. Proteinase K is then added to the mixture to degrade the protein in the sample.

What is step 6 of ChIP?

DNA purification and PCR analysis

What happens in step 6?

DNA is purified to remove protein and salt impurities from the mixture to facilitate PCR analysis of ChIP experiment. Primers specific to candidate gene promoter elements (e.g. BMS-1) are utilised in PCR to examine binding of transcription factors to regulatory elements. Enrichment of transcription factor binding is compared to control IgG