Elsevier

Methods

Volume 33, Issue 1, May 2004, Pages 81-85
Methods

Use of bifunctional cross-linking reagents in mapping genomic distribution of chromatin remodeling complexes

https://doi.org/10.1016/j.ymeth.2003.10.022Get rights and content

Abstract

Chromatin remodeling complexes consist of multiple subunits, some of which are in intimate contact with DNA while others are not. The ability to effectively cross-link proteins to DNA with formaldehyde is impacted by the average distance between the two species. Productive cross-linking of proteins not in direct contact with DNA is greatly facilitated by the inclusion of an initial cross-linking reaction using bifunctional imidoester cross-linking reagents.

Introduction

The determination of sites of interaction of nuclear proteins with specific DNA sequences has become a matter of great interest. Multiple techniques can be utilized to derive such information including immunolocalization [1], various types of nuclease digestion [2], [3], and in vivo footprinting [4]. In addition, cross-linking followed by immunoprecipitation has evolved as a powerful tool for mapping protein–DNA interactions [5], [6]. Widely used protocols rely on chemical or physical cross-linking reagents to preserve native nuclear structures for subsequent biochemical and molecular analysis. Here, we describe a simple modification of a commonly used chemical cross-linking protocol that facilitates determination of genomic sites of occupancy of chromatin remodeling complex subunits not in intimate contact with DNA.

General characteristics of cross-linking reagents of interest to mapping protein–DNA interactions include preservation of native structures, sufficient stability to permit biochemical and/or molecular analysis, reversibility, activity under physiologic conditions, ability to freely enter cells, and resolution. Formaldehyde is perhaps the most widely used chemical cross-linker for preservation of nuclear structure prior to biochemical analysis of protein–DNA interactions [7]. Formaldehyde is very reactive towards amino and imino groups such as the side chains of lysine and arginine and leads to the formation of a Schiff base (see Fig. 1). This Schiff base can then react with a second amine, leading to a covalent cross-link between two amino groups. This chemistry is reversible and conditions have been described to reverse protein–DNA cross-links as well as protein–protein cross-links [7], [8]. Importantly, formaldehyde is a high resolution reagent that acts across a short distance—2 Å.

In general, chromatin remodeling complexes are large macromolecular species composed of many different subunits [9]. While it is clear that a subset of the protein subunits of these complexes is in intimate contact with DNA, it is highly likely that others are not. In the case of a protein not in intimate contact with DNA, the probability of a successful direct cross-link to DNA with high resolution reagents such as formaldehyde is low. The retention of such subunits during biochemical analysis would then require maintenance of native structure, conditions not frequently utilized in common chromatin immunoprecipitation protocols [10]. An adjunct cross-linking reagent can be used to facilitate retention of chromatin remodeling complex integrity. We [11] and others [12] have successfully used bifunctional imidoester cross-linkers with longer effective distance between functional groups as an adjunct to standard formaldehyde cross-linking. Importantly, these reagents freely permeate cells making them ideal candidates for chromatin cross-linking. Reagents successfully used for ChIP studies in this class include dimethyl 3,3-dithiobispropionimidate (DTBP) and dimethyl adipimidate (DMA) with resolution of 11.9 and 8.6 Å, respectively [11], [12].

The imidoester class of bifunctional cross-linking agents is highly reactive towards primary amines. Reaction pH is critical, alkaline conditions (pH 8–10) favor amidine formation. The reactivity of alkyl amines (such as the ε amino group of lysine) towards these reagents differs substantially from aromatic amines (such as DNA bases). Thus, these reagents efficiently cross-link proteins to other proteins (see Fig. 1) but are not effective as protein–DNA cross-linkers. They are typically used in conjunction with a standard protein–DNA cross-linking reagent such as formaldehyde.

Section snippets

Cross-linking adherent mammalian cells

Here, we present a simple protocol for the application of the two-step cross-linking protocol to adherent mammalian cells. The application of very similar techniques to yeast cells has been recently outlined by Kurdistani and Grunstein [10].

Materials required

  • •

    Lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris–HCl, pH 8.0, and 1 mM PMSF).

  • •

    Dilution buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl, pH 8.0, and 167 mM NaCl).

  • •

    Sonicator.

Protocol

  • 1.

    Add lysis buffer (200 μl per 106 cells) directly to cells on plate, scrape with rubber policeman, and incubate for 10 min on ice.

  • 2.

    Collect lysed cells in microfuge tube. Use cell scraper to remove all cells and lysis buffer.

  • 3.

    Sonicate on ice. We use a Branson Sonicator with microtip and sonicate for 15 s (output control=4, 50% duty

Materials required

  • •

    Dilution buffer (1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl, pH 8.0, 167 mM NaCl, and 1 mM PMSF).

  • •

    Protein A–agarose/salmon sperm DNA (Upstate Biotech).

  • •

    Wash buffer 1 (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 2 mM EDTA, and 0.1% SDS).

  • •

    Wash buffer 2 (20 mM Tris–HCl, pH 8.0, 500 mM NaCl, 1% Triton X-100, 2 mM EDTA, and 0.1% SDS).

  • •

    Wash buffer 3 (10 mM Tris–HCl, pH 8.0, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, and 1 mM EDTA).

  • •

    TE buffer (10 mM Tris–HCl, pH 7.5, 1 mM EDTA).

  • •

    Elution buffer (1% SDS,

Application of the two-step cross-linking protocol—the Mi-2/NuRD complex

The vertebrate Mi-2/NuRD complex is a multisubunit chromatin remodeling complex [13], [14]. Determination of sites of interaction of this complex with genomic DNA is of obvious interest. While it is likely that some subunits of this complex are in direct contact with DNA, others are not. We have recently determined a site of action of the Mi-2 complex in breast cancer cell lines [11]. Cross-linking of human breast cancer cells was performed as described here. We used both standard formaldehyde

Conclusions

This protocol has been successfully applied to determination of the distribution of the human Mi-2/NuRD complex in human cells [11]. A variation has been employed by the Grunstein laboratory to isolate genomic DNA for microarray analysis [12]. The number of available bifunctional cross-linking reagents is large. These reagents differ in important fundamental properties. Their application to the problems of mapping protein–DNA interactions in native chromatin promises to continue to emerge as an

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