Mapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation

doi:10.1016/S0968-0004(99)01535-2

Trends in Biochemical Sciences

Volume 25, Issue 3, 1 March 2000, Pages 99-104

https://doi.org/10.1016/S0968-0004(99)01535-2 Get rights and content

Abstract

Gene regulation is a complex process. Numerous factors appear to be required for the accurate temporal and spatial regulation of each gene. Often these factors are assembled into multiprotein complexes, contributing to specific gene regulation events. Understanding how all these factors are organized in the chromosome and how their function is regulated in vivo is a challenging task. One of the most useful techniques for studying this level of gene regulation is the in vivo fixation by formaldehyde crosslinking of proteins to proteins and proteins to DNA, followed by immunoprecipitation of the fixed material.

Section snippets

The chemistry of formaldehyde crosslinking: the advantage of de-crosslinking

Formaldehyde is a tight (2 Å) crosslinking agent that efficiently produces both protein–nucleic acid and protein–protein crosslinks in vivo. Formaldehyde is a very reactive dipolar compound in which the carbon atom acts as a nucleophilic centre. Amino and imino groups of amino acids (lysines, arginines and histidines) and of DNA (primarily adenines and cytosines) readily react with formaldehyde leading to the formation of a Schiff base. This intermediate can further react with a second amino

Extent of crosslinking: finding the right balance

Efficient fixation of a protein to chromatin in vivo is crucial for the X-CHIP technique. The extent of crosslinking is probably the most important parameter. Two major problems concerning the subsequent immunoprecipitation step should be taken into account: first, an excess of crosslinking can result in the loss of material or reduced antigen availability in chromatin, or both, and second, the relative sensitivity of the antigen epitopes to formaldehyde.

Crosslinking times range between 10

Antibodies and immunoprecipitation conditions

In the X-CHIP assay, the immunoprecipitation step requires highly stringent conditions. A buffer, called RIPA, of intermediate ionic strength and containing a combination of denaturing and non-denaturing detergents [Triton, sodiumdeoxycholate and sodium dodecyl sulphate (SDS)], is used to ensure solubility of chromatin6, 9. The use of affinity-purified antibodies is highly recommended, and polyclonal antibodies are preferred to monoclonals to avoid potential epitope masking problems in

The use of a caesium chloride gradient

In the original protocol by Varshavsky and co-workers designed for Drosophila tissue culture cells, the purification of crosslinked chromatin using caesium cloride (CsCl) gradients was described⁶. This procedure is rather time-consuming as it involves 72 hours of ultracentrifugation6, 10. An alternative method is the sonication of fixed material in high-detergent buffer and adjustment to immunoprecipitation-compatible conditions by simple dilution¹¹. This procedure has been successfully applied

Analysis by PCR of X-CHIP DNA

Michael Grunstein’s team has described a method for the analysis of X-CHIP products based on a quantitative PCR approach12, 13. After DNA purification from immunoprecipitated chromatin, the enrichment of specific genomic fragments corresponding to specific protein-binding sites are scored by quantitative PCR.

Specific pairs of primers covering contiguous fragments of the genomic region of interest are designed. An empirically defined number of amplification cycles is used and the amplified

Southern-blot analysis of X-CHIP DNA

A different approach utilizes the X-CHIP DNA as a probe in Southern-blot analysis9, 10. The advantage of this ‘one-step’ analysis is the rapid identification of binding sites within large genomic regions without relying on multiple PCR reactions. The resolution with this method is also ∼300 bp. In this type of analysis, the genomic fraction obtained by immunoprecipitation, containing virtually all the in vivo binding sites of a given chromatin protein, is radiolabelled and used as a probe

Future applications and conclusions: identification of target genes

The ability to determine the in vivo binding sites of chromosomal proteins by X-CHIP could be of interest for the identification of target genes of specific transcription factors. X-CHIP has been used successfully in mammals for the identification of target genes of the HoxC8 and Oct4 proteins5, 25. In the case of HoxC8, fixed chromatin from whole-mouse spinal cord was used for X-CHIP and the specifically enriched DNA subcloned in l-vectors. Each subclone was then used as a probe to screen a

Acknowledgements

I would like to thank Andreas Hecht and Michael Grunstein for providing their original data, Bhavin Parekh and Tom Maniatis for sharing technical information prior to publication, Renato Paro and members of his laboratory for having shared the navigation in the X-CHIP hyperspace, and Paul Orban for proofreading the manuscript. This work is supported by the Associazione Italiana Ricerca sul Cancro and Telethon.

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Trends in Biochemical Sciences

TechniquesMapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation

Abstract

Section snippets

The chemistry of formaldehyde crosslinking: the advantage of de-crosslinking

Extent of crosslinking: finding the right balance

Antibodies and immunoprecipitation conditions

The use of a caesium chloride gradient

Analysis by PCR of X-CHIP DNA

Southern-blot analysis of X-CHIP DNA

Future applications and conclusions: identification of target genes

Acknowledgements

Cell

Cell

Methods

Cell

Anal. Biochem.

Mol. Cell

Cell

Cell

Cell

Cell

Cell

Formaldehyde as a probe of DNA structure. I. Reaction with exocyclic amino groups of DNA bases

Biochemistry

Formaldehyde as a probe of DNA structure. II. Reaction with endocyclic imino groups of DNA bases

Biochemistry

Formaldehyde-mediated DNA-protein crosslinking: a probe for in vivo chromatin structures

Proc. Natl. Acad. Sci. U. S. A.

New POU dimer configuration mediates antagonistic control of an osteopontin preimplantaion enhancer by Oct-4 and Sox-2

Genes Dev.

Transcriptional silencing in yeast is associated with reduced nucleosome acetylation

Genes Dev.

Techniques
Mapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation