Background

Studies aiming to elucidate the molecular underpinnings of genomic processes influenced by chromatin have utilized nuclease sensitivity to assess the accessibility of chromatin. This is a powerful concept for interrogating the regulation and timing of chromatin conformational changes that contribute to cellular processes such as DNA replication and transcription. For example, it has been shown that chromatin is more sensitive to micrococcal nuclease at two distinct sites within the regions encompassing origins of DNA replication in Chinese hamster ovary cells during G₁ versus S-phase, suggesting that chromatin is more accessible at these origins in G₁ phase (Pemov et al., 1998). However, the approach used in this latter study required laborious techniques, Southern blotting, large-scale and complex genomic DNA collections and restriction digests, and cumbersome gel-electrophoresis and membrane-transfer methods. In addition, the ability to interpret the results was limited, and only allowed one to observe changes to a single site in a larger locus, with no other changes in nuclease (chromatin) accessibility being detectable throughout the region. Older more classical techniques, such as nuclease-protection footprinting assays, can give more insight into specific loci in terms of chromatin changes. For example, the positioning or rearranging of nucleosomes can be detected by such techniques in a small region. However, these footprinting techniques also require laborious methods by the investigator, and are not amenable to asking questions regarding large-scale changes to chromatin accessibility in specific regions of interest in the genome.

To overcome many of the limitations of older techniques, our group developed a more simple and efficient method of detecting differences in large-scale chromatin accessibility for any site in the genome that an investigator wishes to assess. Our approach relies on the accessibility of DNase I to perform an initial limited digestion of genomic DNA in the context of chromatin prior to using such DNA as a substrate for an efficient quantitative real-time PCR (qPCR) analysis. No radioactivity or cumbersome gel-electrophoretic methods are required, and isolation of chromatin/DNA for nuclease digestion is straightforward. Analysis is not complicated, and the results are obtained using the rationale that the more DNase I can pre-digest the chromatin/DNA sample, the less substrate DNA there will be for qPCR relative to chromatin/DNA not exposed to DNase I (total uncut DNA). Less substrate DNA due to DNAse I digestion will require more PCR cycles to amplify, and this will be interpreted as indicating that the chromatin was more accessible for nuclease digestion. The investigator can manipulate the cells using various methods prior to isolating chromatin, allowing quick and efficient quantitative comparisons of chromatin accessibility changes to a specific region under different experimental conditions. The main limitations of our approach are knowing the genomic DNA sequences of the region under investigation, for primer design, and access to a real-time PCR machine.

In practice, our group has used this DNase I chromatin accessibility technique to assess changes at the human Lamin B2 origin of DNA replication in multiple studies. We demonstrated that chromatin at this origin was less accessible to DNase I (more condensed) in S phase (Wong et al., 2010) or in the absence of endogenous Myc protein specifically in late-G₁ (Nepon-Sixt et al., 2019), since the qPCR signal from the Lamin B2 origin increased (required fewer cycles) under both of these conditions. While this assay has been utilized to determine chromatin accessibility at a specific origin of mammalian DNA replication under various conditions, chromatin accessibility at other genomic sites (e.g., promoters) can be evaluated by using suitable primers during qPCR designed against other sites of interest.