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Genome-wide quantification of transcription factor binding at single-DNA-molecule resolution using methyl-transferase
Rozemarijn W D Kleinendorst1, Guido Barzaghi1,2, Mike L Smith1
1European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Heidelberg, Germany.
Nature Protocols
|November 13, 2021
Summary
We developed single-molecule footprinting to map multiple proteins, including transcription factors and RNA polymerase II, on DNA. This method reveals how these factors cooperate or antagonize to control gene expression.
Area of Science:
- Molecular Biology
- Genomics
- Epigenetics
Background:
- Precise gene expression control relies on the interplay of various factors at cis-regulatory elements.
- Understanding the simultaneous occupancy of these factors on DNA is crucial for deciphering gene regulation.
Purpose of the Study:
- To introduce a novel technique, single-molecule footprinting, for simultaneously resolving the occupancy of multiple proteins on single DNA molecules genome-wide.
- To enable the analysis of cooperative and antagonistic interactions among transcription factors in regulating transcription.
Main Methods:
- The technique combines cytosine methyltransferases for DNA footprinting with bisulfite sequencing to map protein binding patterns.
- Permeabilized nuclei are incubated with methyltransferases, followed by DNA extraction and library preparation for high-throughput sequencing.
- Analysis is facilitated by a dedicated R package and requires high-performance computing.
Main Results:
- Single-molecule footprinting allows simultaneous resolution of multiple protein occupancies, including transcription factors, RNA polymerase II, and nucleosomes, on individual DNA molecules.
- The method provides genome-wide insights into transcription factor binding patterns at cis-regulatory elements.
- The protocol is efficient, completable within 4-5 days, with analysis in 2 days.
Conclusions:
- Single-molecule footprinting offers a powerful approach to study the complex interactions of regulatory proteins at the DNA level.
- This technique advances our understanding of how transcription factors cooperate and antagonize to precisely control gene expression.
- The method is accessible to laboratories with standard high-throughput sequencing capabilities.
