Formaldehyde crosslinking: a tool for the study of chromatin complexes

Abstract

Formaldehyde has been used for decades to probe macromolecular structure and function and to trap complexes, cells, and tissues for further analysis. Formaldehyde crosslinking is routinely employed for detection and quantification of protein-DNA interactions, interactions between chromatin proteins, and interactions between distal segments of the chromatin fiber. Despite widespread use and a rich biochemical literature, important aspects of formaldehyde behavior in cells have not been well described. Here, we highlight features of formaldehyde chemistry relevant to its use in analyses of chromatin complexes, focusing on how its properties may influence studies of chromatin structure and function.

Keywords: chromatin immunoprecipitation (ChIP); chromatin structure; formaldehyde chemistry; nucleic acid chemistry; protein cross-linking; transcription factor.

FIGURE 1.

Graphic depicting the main aspects of formaldehyde reactivity in cells. The dashed arc represents cell or nuclear membranes, which are thought to be highly permeable to formaldehyde (red circles). The thick black curved line represents DNA, shown assembled as nucleosomes (light gray circles). A chromatin-interacting factor is schematized in cyan, with other partner proteins shown in dark blue and purple. Small molecules such as glycine and Tris that react with formaldehyde and can therefore quench reactivity with cellular constituents are shown as green circles. Formaldehyde can crosslink macromolecules together as well as modify exposed groups on macromolecules, forming a product species potentially stabilized by reactivity with a quencher. Quenchers are ordinarily added to the extracellular milieu and may exert their main effects outside the cell.

FIGURE 2.

Chemical reactions occurring during formaldehyde crosslinking of biomolecules. Formaldehyde crosslinking of biomolecules occurs in two steps. First, formaldehyde reacts with a relatively strong nucleophile, most commonly a lysine ϵ-amino group from a protein. This reaction forms a methylol intermediate that can lose water to yield a Schiff base (an imine). Second, the Schiff base reacts with another nucleophile, possibly an amino group of a DNA base, to generate a crosslinked product. This second nucleophile might also be from another protein, the same protein as the first nucleophile, a quencher molecule, or another endogenous small molecule, and therefore a protein-DNA crosslink is only one of many possible products. All of the reactions in this two-step process are reversible, which is a key feature of formaldehyde crosslinking for chromatin capture. A specific example of a protein-DNA crosslink is shown. The atoms are color coded to match those of Fig. 1: cyan, protein; red, formaldehyde; and black, DNA.

FIGURE 3.

Formaldehyde quenching reactions with glycine and Tris, the two most common quenchers. The chemical reactions are analogous to those shown in Fig. 2 with the amino group of glycine or Tris acting as the primary nucleophile. The Schiff base formed from glycine may or may not react with a second nucleophile, but regardless, the crosslinking between macromolecules has been quenched. The Tris molecule has readily available second nucleophiles (hydroxyl groups) that create stable intramolecular five-membered rings. It is also possible for Tris to react with two formaldehyde molecules, leading to the final product shown. The propensity for Tris to form these stable intramolecular products likely allows it to scavenge formaldehyde from other molecules and thereby facilitate crosslink reversal. The atoms are color-coded: green, quencher; red, formaldehyde; brown, miscellaneous nucleophile.

FIGURE 4.

Potential effects of formaldehyde in mediating formation of higher order chromatin structures. The black wavy lines denote chromatin fibers, which may become a crosslinked meshwork in the presence of formaldehyde (red circles). The formation of these potentially confounding structures may or may not be mediated by physiologically relevant higher order interactions captured by crosslinking (dashed gray rectangle). Such a meshwork may define localized neighborhoods in the nucleus that trap proteins (cyan) that may or may not interact specifically with nearby DNA sequences in an unperturbed cell.