Second-generation method for analysis of chromatin binding with formaldehyde–cross-linking kinetics

Abstract

Formaldehyde-cross-linking underpins many of the most commonly used experimental approaches in the chromatin field, especially in capturing site-specific protein-DNA interactions. Extending such assays to assess the stability and binding kinetics of protein-DNA interactions is more challenging, requiring absolute measurements with a relatively high degree of physical precision. We previously described an experimental framework called the cross-linking kinetics (CLK) assay, which uses time-dependent formaldehyde-cross-linking data to extract kinetic parameters of chromatin binding. Many aspects of formaldehyde behavior in cells are unknown or undocumented, however, and could potentially affect CLK data analyses. Here, we report biochemical results that better define the properties of formaldehyde-cross-linking in budding yeast cells. These results have the potential to inform interpretations of "standard" chromatin assays, including chromatin immunoprecipitation. Moreover, the chemical complexity we uncovered resulted in the development of an improved method for measuring binding kinetics with the CLK approach. Optimum conditions included an increased formaldehyde concentration and more robust glycine-quench conditions. Notably, we observed that formaldehyde-cross-linking rates can vary dramatically for different protein-DNA interactions Some interactions were cross-linked much faster than the macromolecular interactions, making them suitable for kinetic analysis. For other interactions, we found the cross-linking reaction occurred on the same time scale or slower than binding dynamics; for these interactions, it was sometimes possible to compute the equilibrium-binding constant but not binding on- and off-rates. This improved method yields more accurate binding kinetics estimates on the minute time scale.

An Improved Method for Measuring Chromatin-binding Dynamics Using Time-dependent Formaldehyde Crosslinking

“…In Competition ChIP (CC), expression of a differentially tagged isoform of the TF of interest is induced and the relative levels of the constitutive and induced forms of the TF are monitored over time, yielding binding kinetic information through measurements of TF turnover at the sites of interest (van Werven et al , 2009; Lickwar et al , 2013). With advancements in modeling of CC data, residence times as short as 1.3 min have been measured (Zaidi et al , 2017). Relative dynamic measurements have also been made by conditional depletion of TFs from the nucleus using the Anchor Away technique (Haruki et al , 2008; Grimaldi et al , 2014), although specific mathematical models of the process have not yet been reported.…”

“…The first iteration of the CLK assay used ‘standard’ crosslinking and quenching conditions (1% formaldehyde and 250 mM glycine, respectively). Additional work has recently yielded experimental conditions that increase the crosslinking rate and improve quenching of the crosslinking reaction (Zaidi et al , 2017). These new conditions have resulted in a more robust method and the ability to model and analyze crosslinking kinetic data with more reliability and confidence.…”

“…Yeast strains, wild type (WT) and overexpression (OE) for each TF of interest (see Poorey et al , 2013 and Zaidi et al , 2017 for strain lists)…”

“…The OE strain can be engineered by introducing into cells an additional copy of the TF gene on a plasmid or by integrating into the genome under control of the native promoter or an appropriate heterologous promoter. If the TF functions as a stable biochemical entity with more than one type of subunit, the OE strain ought to be engineered to drive balanced expression of each subunit, as for example, was done for the analysis of TFIIE (Zaidi et al , 2017). For a detailed description of OE strain and plasmid construction, see Poorey et al , 2013, Zaidi et al , 2017.…”

“…As mentioned above, the experimental conditions required for analysis of a particular TF should be validated and may need to be optimized as described in Procedure D and E. This section describes the basic CLKv2 procedure as we optimized and recently described (Zaidi et al , 2017). …”