Measuring the Formaldehyde Protein–DNA Cross-Link Reversal Rate

Abstract

Protein–DNA binding interactions play critical roles in important cellular processes such as gene expression, cell division, and chromosomal organization. Techniques to identify and characterize these interactions often utilize formaldehyde cross-linking for stabilization of the complexes. Advantages of formaldehyde as a cross-linking reagent include cell permeability, relatively fast cross-linking kinetics, and short cross-linker length. In addition, formaldehyde cross-links are reversible, which has the advantage of allowing complexes to be dissociated if desired but may also present a problem if undesired dissociation occurs in the course of an experiment. While the kinetics of formaldehyde cross-link formation have been well-established in numerous studies, there have been no reports of the rate of cross-link dissociation, even though it is clearly a critical variable when developing a biochemical protocol involving formaldehyde cross-linking. We present here a method for measurement of the rate of formaldehyde cross-link reversal based upon the Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) procedure and use it to determine the rate of cross-link reversal for cross-linked protein–DNA complexes from yeast cell lysate. The half-life of the protein–DNA cross-links varies from 179 h at 4 °C to 11.3 h at 47 °C, with a rate that increases exponentially with temperature and is independent of salt concentration.

Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes

“…The reversibility of formaldehyde crosslinking has been explored in some detail in an effort to recover proteins from fixed tissue and cell samples (84,85). The temperature and salt concentration dependence of the formaldehyde crosslink reversal rate has been established, revealing a crosslink half-life consistent with the estimate of crosslink half-life in cells (tens of hours at 37°C) (86). That study also quantitatively showed the extent to which heat can increase the crosslink reversal rate.…”

“…This explains why proteins and DNA isolated from formaldehyde-treated cells appear unmodified in general (14,83,92). Within minutes of formaldehyde incubation, there is very little detectable free DNA (Ͻ10%) (36,86), and crosslinking appears to occur uniformly along DNA as well (14).…”

Sequence Artifacts in DNA from Formalin-Fixed Tissues: Causes and Strategies for Minimization

“…Formaldehyde-induced crosslinks are reversible by heat treatment (47 ). The reversal rate of formaldehyde crosslinks is closely dependent on the temperature and pH of the buffer solution (48,49 ). The half-life of formaldehyde crosslinks is inversely correlated with temperature (48 ).…”

“…The reversal rate of formaldehyde crosslinks is closely dependent on the temperature and pH of the buffer solution (48,49 ). The half-life of formaldehyde crosslinks is inversely correlated with temperature (48 ). High-temperature heating methods, usually at Ͼ90°C, have been shown to be effective not only for the yield of DNA (50 ), but also for the yield of amplifiable templates from FFPE tissues (49,51 ).…”

Analysis of chromatin binding dynamics using the crosslinking kinetics (CLK) method

“…The derivation of the model begins with a simple reaction describing the process of TF binding to DNA, followed by crosslinking: $$\text{italicunbound}TF\underset{k_d}{\overset{k_a}{\rightleftharpoons }}\text{italicbound}TF\stackrel{k_{xl}}{\to }\text{italiccrosslinked}TF$$ In this reaction, the reversible binding of a TF to DNA is governed by the rate constants k a and k d , and formaldehyde crosslinking occurs at a rate defined by k xl and is presumed to be irreversible under our experimental conditions. Formaldehyde crosslinking has been known for decades to be very stable [22] and irreversibility of the crosslinking reaction under the conditions described here is supported by direct measurements of the formaldehyde-mediated protein-DNA crosslink reversal rate [23]; discussed more below. The derived CLK model is very general, with the only assumptions being that (1) the ChIP signal is crosslinking dependent, (2) DNA binding by the TF precedes crosslinking, (3) the TF is in excess over chromatin sites and (4) that the unbound TF pool is not substantially depleted by nonspecific inactivation by formaldehyde.…”