Unraveling Gene Transcription: Exploring Chromatin Accessibility with ATAC-Seq

Gene transcription requires the unraveling of a higher structural part of the DNA. This part of the chromatin is called accessible chromatin region and this process is achieved mainly by the modification of chromosomal histones (especially acetylation). The property of chromatin is called chromatin accessibility, so that chromatin accessibility reflects the state of binding of regulatory factors to open chromatin, which is closely related to transcriptional regulation. ATAC-seq technique uses Tn5 transposase to cleave regions of DNA that are not protected by binding proteins, and is used to analyze chromatin openness and to determine dynamic changes in chromatin structure.

Principle of ATAC-Seq

By cleaving the open nuclear chromatin region at a specific space-time by transposase, we obtain the regulatory sequences of all active transcripts in the genome at that space-time, and then discover the key regulatory transcription factors by mining the potential binding transcription factors at these open sites through clustering analysis, combined with gene expression level data.

Advantages of ATAC-Seq

• High sensitivity, low cell starting volume of 50,000 cells per sample
• Simplified experimental steps, good reproducibility and high success rate
• simultaneously reveals the genomic location of open chromatin, DNA binding protein and transcriptional binding site interactions
• ATAC-seq is now the technique of choice for studying chromatin accessibility

Workflow of ATAC-Seq

• Using Tn5 transposase, a transposable complex carrying a sequencing tag is added to the nucleus and subsequently amplified by PCR using the tag to identify open chromatin.
• The fragmented DNA is purified, a sequencing library is constructed, and the samples are sequenced using NGS sequencing technology.
• After comparing the sequenced fragments to the reference genome, the open chromatin regions can be identified.

Bioinformatic Analysis of ATAC-Seq

• Fastq quality control. Remove low quality and contaminated splice sequences
• bowtie2 alignment. Alignment of filtered reads to the reference genome
• Post-comparison QC
• Peak calling
• Peak annotation
• Motif analysis
• Footprint analysis

What is the difference between ATAC-seq and ChIP-seq?

ChIP-Seq generally designs antibodies to do ChIP experiments to pull DNA based on the target transcription factor to verify whether the transcription factor of interest interacts with DNA.

Whereas ATAC-Seq generally detects chromatin openness on a genome-wide scale and can get genome-wide information on the possible binding sites of proteins, using this technical approach in combination with other methods is trying to screen the regulatory factors of interest.

How does ATAC-seq study accessible chromatin regions (ACRs)?

The common chromatin open regions are mainly the promoter upstream of the gene and the distal regulatory elements such as enhancers and silencers. The promoter is the DNA region near the transcription start site (TSS), which contains the transcription factor binding site (TFBS), so the transcription factor can bind to the TFBS on the promoter and recruit RNA polymerase to transcribe the gene. The TSS contains a transcription factor binding site (TFBS), so the transcription factor can bind to the TFBS on the promoter and recruit RNA polymerase to transcribe the gene. Enhancers are generally located in the 1 Mb DNA region downstream or upstream of the promoter, and when transcription factors bind to enhancers and make contact with the promoter region, they can promote gene transcription. On the contrary, silencers reduce or repress the expression of genes.

ATAC-seq can help identify promoter regions, potential enhancers or silencers, that is, the peaks in ATAC-seq, which are often promoter and enhancer sequences, as well as some trans-regulatory factors binding sites.

Applications of ATAC-seq

1. Identification of transcription factors involved in gene regulation in combination with motif analysis. The chromatin open region captured by ATAC is generally upstream and downstream of the part of the DNA sequence being transcribed, so that the enriched sequences can be combined with motif analysis to identify which transcription factors are involved in gene expression regulation, including the study of the promoter region where the transcription factors bind.

1. Identification of target genes and functional elements regulated by transcription factors.
2. To analyze in conjunction with super-enhancer identification to clarify the range of active super-enhancers.
3. Better understanding the mechanisms of gene regulation and cellular response to drugs or diseases.
4. Identify transcription factors that drive cell fate, disease or response-related factors, such as cancer.

References:

1. Gehrke A R, Srivastava M. Assessing Chromatin Accessibility During WBR in Acoels[M]//Whole-Body Regeneration: Methods and Protocols. New York, NY: Springer US, 2022: 549-561.
2. Yan F, Powell D R, Curtis D J, et al. From reads to insight: a hitchhiker's guide to ATAC-seq data analysis. Genome biology, 2020, 21: 1-16.
3. Chaitankar V, Karakülah G, Ratnapriya R, et al. Next generation sequencing technology and genome wide data analysis: Perspectives for retinal research. Progress in retinal and eye research, 2016, 55: 1-31.