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Related Experiment Video

Updated: Feb 11, 2026

Formaldehyde-assisted Isolation of Regulatory Elements to Measure Chromatin Accessibility in Mammalian Cells
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An Improved Method for Measuring Chromatin-binding Dynamics Using Time-dependent Formaldehyde Crosslinking.

Elizabeth A Hoffman1, Hussain Zaidi1, Savera J Shetty1

  • 1Department of Biochemistry and Molecular Genetics, University of Virginia Health System, Charlottesville, VA, USA.

Bio-Protocol
|April 24, 2018
PubMed
Summary

We developed crosslinking kinetics (CLK) analysis to measure dynamic transcription factor (TF)-DNA interactions. This method quantizes TF binding kinetics, revealing occupancy and residence times for improved in vivo studies.

Keywords:
Chromatin immunoprecipitation (ChIP)Chromatin structureFormaldehyde chemistryNucleic acid chemistryProtein cross-linkingProtein dynamicTranscription factor

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Area of Science:

  • Molecular Biology
  • Biophysics
  • Genomics

Background:

  • Formaldehyde crosslinking with chromatin immunoprecipitation (ChIP) is standard for mapping transcription factor (TF)-DNA interactions in vivo.
  • Current ChIP methods lack the ability to provide unambiguous data on the dynamic properties of these interactions.
  • Understanding TF-DNA interaction dynamics is crucial for deciphering gene regulation.

Purpose of the Study:

  • To develop and refine a method for estimating TF-DNA binding kinetic parameters from time-dependent crosslinking data.
  • To introduce crosslinking kinetics (CLK) analysis, specifically CLKv2, for quantitative assessment of TF-chromatin interactions.
  • To provide a robust protocol for analyzing TF binding dynamics, applicable to various transcription factors.

Main Methods:

  • Utilized time-dependent formaldehyde crosslinking of yeast cell cultures.
  • Applied mass-action CLK model fitting to ChIP data to extract kinetic parameters (on-rate, off-rate, crosslinking rate).
  • Developed CLKv2 with optimized crosslinking/quenching conditions and systematic modeling procedures.

Main Results:

  • Successfully estimated binding kinetic parameters, including occupancy and residence time, from crosslinking data.
  • Demonstrated the application of CLKv2 analysis to study the binding behavior of TATA-binding protein (TBP) and other TFs.
  • The CLKv2 protocol provides a systematic approach to model different kinetic regimes of TF-chromatin interactions.

Conclusions:

  • Crosslinking kinetics (CLK) analysis, particularly CLKv2, offers a powerful approach to quantitatively assess TF-DNA interaction dynamics.
  • The method provides insights into binding kinetics (on/off rates, occupancy, residence time) not achievable with standard ChIP.
  • The CLKv2 protocol is adaptable for studying TF binding in yeast and potentially other organisms.