Formaldehyde treatment is a popular way to cross-link proteins and DNA during Chromatin Immunoprecipitation (ChIP), an assay that has been widely used to characterize protein-DNA interactions. Now, a team from University of Virginia Health System has enhanced this time-tested formaldehyde fixation approach to understand how fast/slow proteins bind to DNA and the stability of chromatin interactions.
According to the researchers, there is a critical period in timing before the cross-linking is finished that can be exploited for its’ physical time quantitating magic, or as author Ramya Viswanathan explains, “…the time dependence of formaldehyde crosslinking can be used to extract in vivo on- and off-rates for site-specific chromatin interactions.”
Auble and colleagues deployed a modified ChIP assay, with sub-second temporal resolution to demonstrate how “…a regulatory process can shift weakly bound TATA-binding protein to stable promoter interactions, thereby facilitating transcription complex formation.”
The newly generated kinetic data unveiled a new model for a gene regulator that was thought to be understood already, demonstrating how being able to do these kinds of measurements can potentially provide new and unexpected insight into chromatin-based phenomena.
Viswanathan also underscored that their method can be universally applied, remarking that “For almost all of the factors that we studied, the ChIP signal depended on formaldehyde incubation time, and that the dynamics of crosslinking and/or chromatin binding can be detected by the CLK method. Using this, we were able to obtain dynamics of factors interacting with chromatin in second time scale even at single copy genes.”
Viswanathan shared with us her team’s conclusions that “This study sheds light on how the crosslinking reaction can be used to obtain kinetic information for several factors over a ~100 fold dynamic range. In addition, we view the relative simplicity of our model as a notable strength and therefore I can’t see why this method cannot be applied to various fields of biology to define the dynamics of several key regulatory processes.”
Get in synch with your formaldehyde fixation times over at Science, December 2013