Deciphering the Genome Through Chromatin Accessibility Assays
The degree to which regulatory elements, such as transcription factors and other DNA-binding proteins, are accessible to chromatin is critical for a variety of cellular processes, including replication, transcription, and DNA repair. Changes in chromatin accessibility play a pivotal role in determining which genes are actively transcribed or silenced, influencing cellular development, differentiation, proliferation, and responses to environmental cues.
Chromatin accessibility assays can directly measure how alterations in the structure of chromatin affect gene expression. This differs from techniques like chromatin immunoprecipitation sequencing, where these effects are instead extrapolated based on the presence or absence of particular histone post-translational modifications. DNase-seq, FAIRE-seq, and ATAC-seq are methods commonly utilized to assess chromatin accessibility and understand the complex regulatory mechanisms governing gene expression in various biological contexts.
Illustration of the schematic procedures for DNase-seq, FAIRE-seq, and ATAC-seq
When it comes to chromatin accessibility assessment, DNase I hypersensitive site sequencing, or DNase-seq, is considered “the gold standard.” This technique exploits the endonuclease’s preferential cleavage of DNase I hypersensitive sites within open, nucleosome-depleted chromatin regions. Such regions, which are more readily degraded by DNase I treatment and thus less frequently identified during sequencing analysis of constructed DNA libraries, can consequently be deduced. Alternatively, by tweaking the treatment and limiting DNase I digestion, short DNA fragments corresponding to regions of open chromatin structure can be generated, selected, and directly sequenced. Cleavage bias and a high input requirement are some of the major limitations of DNase-seq.
Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE), paired with sequencing, was introduced in succession to DNase-seq. It is based on the differential crosslinking properties of formaldehyde between open chromatin and nucleosomes. FAIRE involves the use of phenol-chloroform extraction to separate nucleosome-bound genomic areas from more accessible regions of sheared chromatin. As crosslinking preserves the in vivo interactions between DNA and associated proteins, the chromatin-accessible regions, which have fewer interactions with proteins, are enriched in the aqueous phase and can thus be isolated. While FAIRE addresses the sequence-specific cleavage bias of DNase I, the method is limited by adequate fixation efficiency and a low signal-to-noise ratio.
The Assay for Transposase-Accessible Chromatin using sequencing, or ATAC-seq, is a more recent advancement in genome-wide chromatin accessibility assessment. With the use of the hyperactive mutant Tn5 transposase, accessible chromatin from within unfixed nuclei is simultaneously fragmented and tagged with sequencing adapters, providing improvements in speed, specificity, and sensitivity from lower starting sample amounts. However, ATAC-seq exhibits significant fragmentation and GC/AT content bias due to the sequence-specific cleavage of the transposase. Additionally, it is disadvantaged by the overrepresentation of mitochondrial DNA (mtDNA) fragments, as the Tn5 enzyme more efficiently transposes mtDNA.
To address some of these shortcomings, EpigenTek has developed the EpiNext™ Chromatin Accessibility Sequencing Fast Kit. This innovative kit offers a more affordable alternative for generating DNA libraries for chromatin accessibility assessment from either cells or tissues and at a fraction of the time (less than 2 hours versus 1-3 days for traditional ATAC-seq). A unique nucleic acid cleavage enzyme mix that exhibits no sequence specificity or fragmentation bias is employed. The extremely rapid protocol time minimizes nuclear damage, better preserves chromatin structure, and eliminates mitochondrial contamination.
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