By: Siwei Zou
Suomennos: Transkriptiotekijöiden jalanjälkianalyysi Fiber-seq-menetelmällä
Transcription factors play a critical role in regulating gene expression by binding to specific regions of DNA. This binding leaves behind “footprints” on the genome—protected regions that give us clues about what genes are being regulated. One of the most advanced methods to study these interactions is Fiber-Seq, which allows us to map transcription factor footprints with high resolution and over long stretches of DNA.
Chromatin Accessibility: The Two Faces of DNA Packaging
Before diving into how Fiber-Seq works, it’s important to understand the structure of chromatin—the material that makes up chromosomes. Human DNA is incredibly long, about 2 meters per cell, yet it fits into a cell nucleus only 5–10 micrometers in diameter. To accomplish this, DNA is wrapped around proteins called histones, forming bead-like structures called nucleosomes. These nucleosomes are packed together tightly to form chromatin, which can either be open and accessible or tightly closed and inaccessible.
The ability of transcription factors to access DNA depends on how loosely or tightly chromatin is packed. In open chromatin, the DNA is more exposed, allowing transcription factors to bind regulatory elements and control gene expression. In contrast, closed chromatin is tightly coiled, making it harder for transcription factors and other regulatory proteins to interact with the DNA. This balance between open and closed chromatin is known as chromatin accessibility, a critical factor in gene regulation.
Fiber-Seq: Mapping Protein Binding at High Resolution
Traditional techniques to study chromatin accessibility, such as ChIP-Seq or ATAC-Seq, break DNA into short fragments before analyzing it. While these methods can provide valuable information, they often miss the larger, continuous picture of chromatin structure and transcription factor binding.
Fiber-Seq overcomes this limitation by using a unique approach. It works by tagging accessible areas of chromatin with a methyltransferase enzyme, which adds chemical markers to the DNA. These markers reveal which regions of the genome are open for transcription factor binding. After tagging, the markers are read by long-read DNA sequencing technologies like PacBio or Nanopore, which can read much larger sections of DNA in one go compared to traditional methods. This allows for the simultaneous detection of transcription factor binding and chromatin architecture across long stretches of the genome.
Transcription Factor Footprints: Revealing Gene Regulation
One of the most valuable applications of Fiber-Seq is in mapping the footprints of transcription factors—those areas of the genome where these proteins bind to regulate gene expression. When transcription factors bind to DNA, they protect that region from being tagged by the methyltransferase, leaving behind a “footprint.” By comparing methylation patterns in regions with and without transcription factor binding, scientists can precisely map these footprints.
For instance, in our lab we explored the chromatin accessibility in colon adenocarcinoma cell line GP5d using Fiber-Seq, and were able to map transcription factor ELF2 binding in the EIF3K gene promoter region. The pre-treatment of ELF2 on nuclei created a footprint by reducing methylation at specific DNA motifs (GGAA**GGAA).
This revealed the exact sites where ELF2 interacts with the genome, providing valuable insight into how gene expression is controlled in these cells. In addition, the footprints of any TF, even unknown, can be revealed by comparing the methylation signal from cells treated with methyltransferase with the methylation signal from cells pre-treated with high salt concentration (up to 0.5 M); the salt treatment should wash away the binding proteins. In this comparison of the genome sequencing data, the footprints should emerge.
The Future of Fiber-Seq in Gene Regulation Research
Fiber-Seq represents a significant advancement in our ability to study transcription factor binding and chromatin structure. Unlike previous methods that focus on fragmented data, Fiber-Seq captures the larger context of chromatin accessibility and protein binding, offering a more comprehensive view of gene regulation.
As technology continues to improve, Fiber-Seq will likely become even more powerful and widely adopted. Its ability to provide high-resolution, long-read insights into chromatin structure and transcription factor binding makes it a promising tool for research in areas such as cancer, development, and epigenetics.
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