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Measuring Arabidopsis chromatin accessibility using DNase I-polymerase chain reaction and DNase I-chip assays - PubMed

Measuring Arabidopsis chromatin accessibility using DNase I-polymerase chain reaction and DNase I-chip assays

Huan Shu et al. Plant Physiol. 2013 Aug.

Abstract

DNA accessibility is an important layer of regulation of DNA-dependent processes. Methods that measure DNA accessibility at local and genome-wide scales have facilitated a rapid increase in the knowledge of chromatin architecture in animal and yeast systems. In contrast, much less is known about chromatin organization in plants. We developed a robust DNase I-polymerase chain reaction (PCR) protocol for the model plant Arabidopsis (Arabidopsis thaliana). DNA accessibility is probed by digesting nuclei with a gradient of DNase I followed by locus-specific PCR. The reduction in PCR product formation along the gradient of increasing DNase I concentrations is used to determine the accessibility of the chromatin DNA. We explain a strategy to calculate the decay constant of such signal reduction as a function of increasing DNase I concentration. This allows describing DNA accessibility using a single variable: the decay constant. We also used the protocol together with AGRONOMICS1 DNA tiling microarrays to establish genome-wide DNase I sensitivity landscapes.

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Figures

Figure 1.
Figure 1.

Outline of the DNase-PCR procedure.

Figure 2.
Figure 2.

Schematic illustration of the digestion procedure. For easier handling and better synchronization, sample aliquots can be transferred using multichannel pipettes. Digestion can be performed taking advantage of PCR tube strips and PCR cyclers. [See online article for color version of this figure.]

Figure 3.
Figure 3.

Principle of using PCR to detect the accessibility of DNA fragments in chromatin. After a limited DNase I digest of native chromatin, more accessible chromatin is cleaved more frequently within a short distance, resulting in fragmentation of the DNA and reduced signals from PCR (right). Inaccessible chromatin is cleaved less frequently; therefore, the DNA remains nearly intact, resulting in strong PCR signals (left). The small arrows represent primer positions. [See online article for color version of this figure.]

Figure 4.
Figure 4.

DNase I-PCR shows differential DNase I sensitivity of chromatin in selected genomic regions. Equal amounts of chromatin and purified DNA were subjected to gradient DNase I treatments. Extracted DNA was used as a template for PCR for two pericentric TE genes (Cinful-like and Ta2), two PcG target genes (AG and FT), and two active genes (ACT7 and GAPDHα). The experiment was replicated three times (rep. 1–3).

Figure 5.
Figure 5.

Fitting curves of the DNase I-PCR results for the selected genomic regions. PCR products as shown in Figure 4 were quantified using an Agilent Bioanalyzer. The measurements were normalized to the input values and fitted to an exponential decay model. The fitted curves for all tested genomic regions are shown for the three replicates (rep. 1–3) for both digested chromatin (top panels) and digested isolated DNA (bottom panels).

Figure 6.
Figure 6.

Distribution of the decay constants of the tested genomic regions on a heat axis. The fitted decay constants were averaged over the three replicates for digested chromatin (blue text) and digested isolated DNA (red text). The results were plotted on a logarithmic axis. Error bars show the

se

of the decay constants. The intercept table shows a summary of the calculated results (top right corner).

Figure 7.
Figure 7.

Quantitative PCR confirmation of the enrichment of hyposensitive over hypersensitive DNA regions in different size fractions after controlled DNase I digestion of nuclei. DNA fractions of four size ranges (more than 17 kb, 6–17 kb, 3–6 kb, and less than 3 kb) were recovered separately from undigested (left three columns) and digested (right three columns) nuclei. Enrichment of hyposensitive (inaccessible) pericentric heterochromatin versus hypersensitive (accessible) euchromatic active genes is represented by the enrichment of signal abundance for the TE gene Ta2 against the active ACT7 gene as measured by quantitative PCR. High levels of enrichment are shown in blue, and depletion is shown in red.

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