In order to analyze chromatin structure biochemically, chromatin is often crosslinked with formaldehyde, and then sheared by sonication or digested by enzymatic reaction. A ChIP assay requires such chromatin, which is usually crosslinked and sheared well for fine structure mapping (10
). Dedon et al.
have applied such chromatin to sedimentation velocity centrifugation on a sucrose gradient, and observed a simple single peak distribution of the chromatin in the gradient (12
). We obtained a similar result showing that such chromatin was retained mostly in the uppermost fraction under our gradient conditions (data not shown). This indicates that chromatin prepared in this manner cannot be fractionated on a sucrose gradient (regardless of its structure), although its buoyant density can be measured as reported previously (13–15
). Non-crosslinked and mildly-sheared chromatin can also be applied to such gradients if an optimum concentration of a cation is included. This strategy has revealed that the β-globin locus in chicken erythrocytes sediments slower than the bulk DNA, suggesting an open state of the chromatin at this active locus (2
). Gilbert et al.
have also used chromatin fractionated by such a method to investigate the relationship between transcriptional activity and chromatin structure in a whole genome (5
). However, the spatial resolution in their strategy is inadequate for the assessment of local chromatin structure.
In this report, chromatin was crosslinked with 0.75% formaldehyde and gently sheared to yield a wide range of DNA (0.1 to >20 kb). After sedimentation on a sucrose gradient, the size-distribution of the DNA purified from each fraction following reverse crosslinking was broadly smeared (C), which is different from that in previous reports using non-crosslinked chromatin (2
). In particular, in our experiments the short fragments (<1 kb) were seen throughout the entire gradient. This suggests that the crosslinking reaction caused some of the small sheared chromatin to be trapped in large particles and to sediment more rapidly toward the bottom of the tube. A microarray analysis of the distribution of a few thousand promoters in the SEVENS assay suggested that those promoters migrating slowly in the gradient tended to be actively transcribing while those trapped in the faster migrating fractions were generally transcriptionally repressed. There are a number of reasons why this pattern of promoter distribution might come about. One technical reason could be the particular concentration of formaldehyde we used for crosslinking (0.75%). When we ran the assay with 1% formaldehyde instead, our four standard genes (Actb
) showed comparable distribution patterns to those seen when 0.75% was used, although only 50–60% of the DNA was recovered. In contrast, with 0.5% formaldehyde the crosslinking was much less and the repressed promoters migrated only to the middle of the gradient despite the continued enrichment of the active promoters in the upper fractions of the gradient (unpublished observations). These observations suggest that the concentration of formaldehyde is critical in determining the fractional distribution of each promoter, but that this is not related to an issue of solubility.
A second possible reason for the promoter distribution pattern is the nucleotide sequences of the promoters themselves, which might have different susceptibilities to crosslinking. This cannot be the sole basis for our results, however, because the same promoter (Il2
) can be found in different fractions of the gradient depending on the activation status of the cell. Nonetheless, the sequences could determine what proteins are bound to the DNA and in particular we considered whether molecules such as polycomb and methyl CpG-binding proteins, which preferentially bind to heterochromatin (16
), might be responsible for good crosslinking and thus direct repressed promoters into the faster sedimenting fractions. This was not the case, however, and in fact those molecules were found mostly in the slower sedimenting fractions of the gradient (Supplementary Figure S3
). In addition, because the structural changes at the Il2
promoter precede recruitment of the RNA polymerase complex and certain transcription factors (unpublished observations), these DNA-binding proteins are also not directly correlated with the structural difference.
A third possible reason is higher-order structures that may be responsible for the crosslinking of small chromatin fragments to larger structures in the nucleus. Some inducible genes can be tethered to other distant loci through a chromatin loop (18
). These contacts could be covalently stabilized by our crosslinking conditions; these might be amenable to further analysis in a chromosome conformation capture (3C) assay. The only proteins we found in large abundance throughout the entire gradient were histones (Supplementary Figure S2
). This suggests that nucleosome structure may be a critical parameter for determining the fractional distribution of the promoters. The number of nucleosomes is known to vary with the transcriptional status of a gene (19
), but we could not correlate the amounts of histone H3 in a ChIP assay with the position of the promoter in the gradient (Supplementary Figure S4
). In addition, our preliminary experiments on the Il2
promoter, which is known to shed a nucleosome on transcriptional activation (7
), showed that eviction of both histone H1 and H3 is an event that occurs after the structural changes seen with our SEVENS assay. Because neighboring nucleosomes can be folded by their direct interaction (20
), we suspect that internucleosomal crosslinking is primarily responsible for the patterns we observe in the gradients. In this scenario, the nucleosomes would be in close proximity on repressed promoters, and thus could be heavily crosslinked, while active promoters would open up and become less amenable to formaldehyde crosslinking. Finally, an enhanced crosslinking mechanism could be working uniquely with promoters in repressed chromatin, which tends to be packaged at very high density in selective regions of the nucleus.
The SEVENS assay we have developed provides a new strategy for examining changes in the chromatin microenvironment at high spatial resolution. Note that the structural changes observed with this assay appear to be distinct from those measured in the nuclease accessibility assay, which occurred more slowly when the Il2
promoter region was observed during T-cell activation (G versus ). This suggests that chromatin remodeling takes place in a multi-step process to facilitate transcriptional activation. The idea that chromatin can assume intermediate higher order structures during gene activation has been previously suggested based on agarose multigel electrophoresis experiments with the mouse mammary tumor virus (MMTV) promoter (22
). Interestingly, the chromatin at the unactivated Il2
promoter also showed an intermediate state in the SEVENS assay (A), with an equal distribution among chromatin fragments of various sizes. It is possible that for the resting state of inducible genes the chromatin is dynamically and spontaneously changing between various open and compact states. In such a model we might be taking a snapshot with the SEVENS assay of the equilibrated chromatin in various cells as a mixture of many configurations. This idea is supported by previous reports showing a dynamic equilibrium state for reconstituted oligonucleosomes (23
), and the ability of the steroid-transcriptionally-activated MMTV promoter to reassume a more compact structure with changes in in vitro
magnesium concentration (22
). Taken together with our recent results for the Ifng
(Interferon-γ) promoter in resting T cells, which showed a similar broad distribution in the sucrose density gradient (data not shown), these intermediate profiles of chromatin structure could reflect a poised state for inducible genes, whose promoter could locally and flexibly unwind in a reversible manner, for example, in a two-start helix model for a 30-nm fiber (25
). Examination of more inducible genes will be required to determine just how general such a poised state could turn out to be.