A new fractionation assay, based on the size of formaldehyde-crosslinked, mildly sheared chromatin, delineates the chromatin structure at promoter regions

Abstract

To explore the higher order structure of transcribable chromatin in vivo, its local configuration was assessed through the accessibility of the chromatin to crosslinking with formaldehyde. The application of crosslinked and mildly sheared chromatin to sedimentation velocity centrifugation followed by size-fractionation of the DNA enabled us to biochemically distinguish between chromatin with heavily versus sparsely crosslinkable structures. The separated fractions showed a good correlation with gene expression profiles. Genes with poor crosslinking around the promoter region were actively transcribed, while transcripts were hardly detected from genes with extensive crosslinking in their promoter regions. For the inducible gene, Il2, the distribution of the promoter shifted in the gradient following T-cell receptor stimulation, consistent with a change in structure at this locus during activation. The kinetics of this switch preceded the chromatin change observed in a DNase I accessibility assay. Thus, this new chromatin fractionation technique has revealed a change in chromatin structure that has not been previously characterized.

Figure 1.

The schema of the SEVENS assay. (A) Chromatin is crosslinked with formaldehyde and sonicated mildly, (B) Chromatin is fractionated by size in a sucrose density gradient, (C) The DNA is purified from each fraction as well as an equal mixture of all the fractions (Sum), and then separated by length in an agarose gel, (D) DNA ranging in length from 0.4 to 0.5 kb (covering 2–3 nucleosomes) is recovered from the gel and (E) A qPCR is performed. The fold enrichment of the region of interest (the amount in each fraction/the amount in the Sum) is calculated. When longer DNA (e.g. 0.8–0.9 kb) was tested, similar results were obtained (data not shown).

Figure 2.

The SEVENS assays for active and repressed control genes. The resting (A and C) and 4 h-activated (B and D) mouse T cells were applied to the SEVENS assay. (A and B) The promoter region of the active genes, Actb (open columns) and Cd3d (closed columns), were enriched in the slowly sedimenting fractions of the gradient and excluded from the faster fractions. (C and D) The promoter region of the repressed genes, Bdnf (open columns) and Adad1 (closed columns), was distributed in a reverse pattern from that of the active genes.

Figure 3.

The expression profiles of the genes selected from the SEVENS assay. (A) RT–PCR was performed for the genes enriched in fraction 2 (shown with bold letters in Table 1). (B) RT–PCR was performed for the genes enriched in fraction 18 (shown with bold letters in Table 2). Because the primers for PCR were designed to anneal to a coding region with a single exon, genomic DNA (g) was utilized as a positive control for the PCR reaction. The plus (+) or minus (–) character means a PCR reaction following cDNA synthesis with or without RT, respectively.

Figure 4.

SEVENS assays for the Il2 promoter region in mouse T cells and liver. (A–F) Chromatin from unactivated (A), 1-h (C), 2-h (D), 3-h (E), or 4-h (F) activated T cells, or liver (B), were run in the SEVENS assay. The fractional distribution of the Il2 promoter (PCR position: −77 to +39) in the sucrose gradient is shown. (G) The fold enrichments of the Il2 promoter in fraction 2 (closed columns) or fraction 18 (open columns) are summarized from the results in unactivated and hourly-activated T cells. (H) The SEVENS assay for unactivated (open columns) or 3-h activated PR-T cells (closed columns) was performed to assess the fold enrichment in fraction 2 of the regions upstream or downstream of the Il2 TSS, which are shown in the lower panel. The numbers denote the relative position to the TSS and the black boxes denote exons.

Figure 5.

DNase I accessibility assay.The amount of each promoter region resistant to DNase I digestion was calculated. A decrease in the resistance at the Il2 promoter (PCR position: −77 to +39) was observed following T-cell activation, suggesting an increase in the accessibility to DNase I. In contrast, both the Actb and Cd3d promoter regions did not show any significant changes during this activation period. The Bdnf promoter was not digested under these experimental conditions (data not shown).