Global survey of chromatin accessibility using DNA microarrays - PubMed (original) (raw)

Global survey of chromatin accessibility using DNA microarrays

M Ryan Weil et al. Genome Res. 2004 Jul.

Abstract

An increasing number of studies indicate a central role for chromatin remodeling in the regulation of gene expression. Current methods for high-resolution studies of the relationship between chromatin accessibility and transcription are low throughput, making a genome-wide study impractical. To enable the simultaneous measurement of the global chromatin accessibility state at the resolution of single genes, we developed the Chromatin Array technique, in which chromatin is separated by its condensation state using either the solubility differences of mono- and oligonucleosomes in specific buffers or controlled DNase I digestion and selection of the large refractory (condensed) DNA fragments. By probing with a comparative genomic hybridization style microarray, we can determine the condensation state of thousands of individual loci and correlate this with transcriptional activity. Applying this technique to the breast tumor model cell line, MCF7, we found that when the condensation is homogeneous in the population of cells, expression is inversely proportional to the level of accessibility and the two methods of accessibility-based target selection correlate well. Using functional annotation and comparative genomic hybridization data, we have begun to decipher the possible biological implications of the relationship between chromatin accessibility and expression.

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Figures

Figure 1

Figure 1

The chromatin solubility assay is used to extract the S1, S2, and P fractions, and the result of that process is depicted as the patterns seen in the gels of the resulting protein and DNA. The protein gel (A) shows the depletion of the H1 histone and the enrichment of nonhistone proteins in the S1 and P fractions. The DNA gel (B) shows the difference in the extent of the digestion between the fractions. The model (C) illustrates the basic concept of the chromatin fractionation assay.

Figure 2

Figure 2

The log scale scatter plot of the co-normalized intensities (sequence abundances) illustrates the relationship between condensation (the ratio of the S2 fraction intensity to total genomic DNA intensity) and the absolute RNA expression. The light gray triangles are Group 2, which has a condensation to expression (C/E) ratio of <0.5. The black squares represent Group 3, which has indeterminate accessibility and a C/E ratio of <2.0 to >0.5. The gray diamonds represent Group 1, which has a C/E ratio of >2.0.

Figure 3

Figure 3

A fragment length selection gel showing the distribution of DNase I-cleaved fragments is used to identify the upper 40% of the total lane intensity to direct the excision of the packing enriched region. The lane digested with the highest concentration of DNase I (64 U) was chosen for its uniform distribution of fragments around the 5-kb median. This section of the gel was excised, and the DNA was recovered for labeling. (M) λ HindIII DNA mass standard.

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