Chromatin structure can strongly facilitate enhancer action over a distance

Efficiency of Enhancer Action Over a Large Distance in Chromatin Does Not Depend on the Level of Unconstrained DNA Supercoiling.

To analyze whether the enhancer can work over a large distance in chromatin, the pLY10 template was used. It contains the glnAp2 promoter that is strongly (>100-fold) activated by NtrC-dependent enhancer over a 2.5-kb distance (9, 18) (Fig. 6A, which is published as supporting information on the PNAS web site). DNA supercoiling can greatly (up to 50-fold) facilitate E–P communication over a large distance on histone-free pLY10, which is the rate-limiting step during distantly activated transcription (9). These observations and the data suggesting presence of unconstrained DNA supercoiling in transcriptionally active eukaryotic chromatin (28, 29) raise the questionof whether unconstrained DNA supercoiling can facilitate enhancer-dependent, distantly activated transcription in chromatin?

Chromatin assembly on negatively supercoiled pLY10 was conducted at various ratios of core histones to DNA (0, 40, 75, and 95% histone loading) by using step dialysis from 2 M NaCl (30). The efficiency of chromatin assembly was monitored by using four assays: quantitative restriction enzyme sensitivity assay (31) (Fig. 6B), analysis of the level of constrained DNA supercoiling in chromatin (32) (Fig. 6C), transmission electron microscopy (Fig. 7, which is published as supporting information on the PNAS web site), and digestion with micrococcal nuclease (Fig. 8, which is published as supporting information on the PNAS web site). The results obtained by different assays are consistent and suggest that the majority of core histones were properly assembled into chromatin and the expected numbers of randomly positioned nucleosome cores were formed on the majority of the pLY10 DNA templates.

The efficiencies of transcription of pLY10 DNA and chromatin templates were compared in a single-round in vitro transcriptional assay (Fig. 1A) (9). The templates were preincubated with all components of the transcription machinery. This incubation results in quantitative binding of inactive NtrC activator to the enhancer and formation of the closed, inactive initiation complex (RPc) at the glnAp2 promoter (15, 18, 33). NtrC activator and the holoenzyme cannot interact with each other because of the lack of NtrC phosphorylation (9, 18). Then ATP was added for a limited time to allow NtrC phosphorylation, E–P communication, and formation of the transcriptionally active open complex (RPo). Subsequent addition of nucleotides and heparin results in completion of initiated transcripts on DNA and chromatin templates and prevents formation of new initiation complexes. The amount of the 401-nt transcripts reflects the rate of formation of open initiation complexes that is equivalent to the rate of functional E–P communication, the rate-limiting step in the whole reaction (9).

Each nucleosome constrains one negative superhelical coil and ≈150 bp DNA (34). Therefore, the level of unconstrained DNA supercoiling remaining after chromatin assembly depends on the initial level of DNA supercoiling (σ) and on the efficiency of the assembly. To evaluate a possible role of DNA supercoiling in enhancer-dependent transcription, chromatin was assembled at 0%, 40%, and 75% histone loading on the pLY10 plasmid having different initial levels of DNA supercoiling σ = −0.055 or −0.065 (Fig. 1B). The levels of unconstrained DNA supercoiling remaining after assembly of 75% chromatin on the templates having initial σ = −0.055 and −0.065 are −0.010 and −0.050, respectively (Fig. 1B). It is assumed that all unconstrained supercoiling remaining after chromatin assembly is concentrated within the linker DNA regions (see Methods). Histone-free pLY10 having σ = −0.050 is transcribed ≈20-fold more efficiently than the same template having σ = −0.010 (data not shown). In contrast, the levels of transcription of 75% chromatin preparations having corresponding levels of unconstrained DNA supercoiling (σ of −0.010 and −0.050) are very similar (Fig. 1C). The data suggest that the efficiency of enhancer-dependent transcription of 75% chromatin does not depend on the level of unconstrained DNA supercoiling. The efficiencies of transcription of 40% chromatin templates are also similar.

The efficiency of transcription of supercoiled pLY10 gradually and moderately decreases in parallel with the increase of histones:DNA ratio (Fig. 1C). Thus formation of 40% and 75% chromatin results in only ≈30–35% and 60% inhibition of transcription, respectively. Much stronger inhibition was expected in cases where chromatin assembly resulted in inhibition of E–P communication. The negative correlation between the extents of transcription inhibition and efficiencies of nucleosome assembly on supercoiled DNA (Fig. 1C) suggests that nucleosome formation most likely blocks binding of the proteins participating in transcription initiation (NtrC activator and/or the holoenzyme) to DNA on a fraction of the templates. As a result, a smaller fraction of templates is used after nucleosome assembly. This interpretation is supported by the data showing direct correlation between the efficiencies of protection of pLY10 DNA from digestion with restriction enzymes and the efficiencies of chromatin assembly (Fig. 6B).

In summary, nucleosome assembly on supercoiled DNA results only in moderate inhibition of transcription initiation; it is unlikely that chromatin assembly strongly decreases the efficiency of E–P communication. The efficiency of transcription of 75% chromatin does not depend on the level of unconstrained negative DNA supercoiling.

Assembly of DNA into Chromatin Can Result in Strong Activation of Enhancer-Dependent Transcription.

To further evaluate the role of chromatin structure and DNA supercoiling in E–P communication over a large distance, chromatin assembled on negatively supercoiled pLY10 plasmid (σ = −0.055) at various histones:DNA ratios was incubated without or with topoisomerase I, which completely relieves unconstrained DNA supercoiling (data not shown). Then the templates were transcribed by using a single-round assay (9, 18) (Fig. 2A).

As expected, the efficiency of transcription of relaxed histone-free pLY10 DNA is very low, primarily because of the low efficiency of E–P communication over 2.5 kb distance (9) (Fig. 2B, lane 2). Remarkably, the efficiency of transcription of relaxed pLY10 was increased up to 5-fold as a result of assembly of 75% chromatin (Fig. 2 B and C). Thus chromatin assembly per se results in a strong positive effect on distantly activated transcription in vitro. Chromatin assembly is unlikely to increase the occupancy of the promoter or enhancer by corresponding proteins. On the contrary, the complete inhibition of transcription after 95% chromatin assembly (Fig. 2B, lanes 7 and 8) matches almost complete protection of the template from the restriction enzymes (Fig. 6B, lane 5) and indicates that chromatin assembly most likely results in a smaller number of the templates accessible to the transcriptional machinery. Therefore, each accessible 75% chromatin template is likely to be used much more efficiently than relaxed DNA. Because E–P communication in the rate-limiting step during transcription of relaxed DNA (9, 12), the data suggest that the efficiency of communication is strongly increased as a result of chromatin assembly.

Transcription of pLY10 template assembled into 40% chromatin remains partially sensitive to DNA supercoiling (Fig. 2B, lanes 3 and 4), most likely because even after chromatin assembly E–P communication is moderately stimulated by remaining negative DNA supercoiling (σ = −0.045, Fig. 1B). Because 40% chromatin templates contain on average six nucleosomes on the 2.5-kb E–P spacer DNA, the data suggest that formation of nucleosome cores does not interfere with E–P communication by the slithering mechanism. In summary, the data suggest that the presence of nucleosome cores does not strongly interfere with E–P communication and can result in strong stimulation of enhancer-dependent, distantly activated transcription of relaxed DNA.

Assembly of DNA into Chromatin Can Strongly Increase the Rate of E–P Communication.

To directly evaluate the rate of E–P communication in chromatin and the possibility that the presence of topoisomerase I affects the outcome of the reaction, transcription of linear pLY10 DNA and chromatin templates was analyzed. Linear pLY10 DNA (Fig. 9A, which is published as supporting information on the PNAS web site) was assembled into 30% or 65% chromatin, or MOCK-assembled. The efficiencies of chromatin assembly were confirmed by analysis of the templates by using the restriction enzyme sensitivity assay (Fig. 9B) and electron microscopy (data not shown). As expected, chromatin assembly on the linear DNA results in a considerable activation of transcription (up to 5-fold in the case of 65% chromatin, Fig. 9 C and D), similar to the effect of chromatin assembly on transcription of the relaxed templates (Fig. 2 B and C).

Next, the rates of E–P communication on negatively supercoiled or linear DNA and 65% linear chromatin pLY10 templates were compared by using the single-round transcription assay (Fig. 3A). In this case, E–P communication was allowed for various time intervals by varying time of incubation with ATP; this resulted in formation of different amounts of the open complexes. Analysis of the rates of E–P communication suggests that, as expected, assembly of 65% chromatin results in utilization of only a fraction of the templates (Fig. 3 B and C). Most likely, the transcriptionally inactive fraction contains nucleosomes that block binding of the proteins participating in transcription initiation (NtrC activator and/or the holoenzyme) to DNA. The rates of E–P communication (t_1/2) on supercoiled DNA and 65% linear chromatin are 4 and 2.5 min, respectively. As expected, the rate of E–P communication on linear DNA was 30 to 50 times lower, as was shown previously (9, 18). Thus, assembly of 65% chromatin results in a >30-fold increase in the rate of E–P communication as compared with relaxed or linear DNA. In summary, assembly of linear DNA into 65% chromatin can greatly facilitate E–P communication over a large distance.

E–P Communication on Chromatin Templates Occurs by a Looping Mechanism.

It has been shown that the NtrC-dependent enhancer can be functionally replaced by curved DNA sequences having no sequence homology to the enhancer (35); this raises a possibility that DNA curved on the surface of nucleosomes could functionally replace the enhancer. However, analysis of transcription of chromatin-assembled pAN6 plasmid containing the same glnAp2 promoter as pLY10 but bearing no enhancer sequences suggests that the presence of the enhancer is absolutely required for the transcription of both DNA and chromatin templates (Fig. 10, which is published as supporting information on the PNAS web site). Thus, DNA curved on the surface of nucleosomes cannot functionally substitute the enhancer.

Further experiments show that transcription of the linear pLY10 template assembled in 65% chromatin is NtrC-, NtrB-, σ⁵⁴-, and ATP-dependent (Fig. 4). Thus, the factor and enhancer requirements for transcription of the histone-free and chromatin-assembled pLY10 templates are identical. Because these factors, on histone-free DNA, participate in direct E–P interaction that is accompanied by formation of a DNA loop (26, 27), a similar looping mechanism most likely operates on the chromatin templates.

Proteins and Protein Complexes.

All proteins and protein complexes were purified as described in ref. 47. Core histones and chicken blood extract containing strong topoisomerase I activity were prepared as described in refs. 48 and 49.

Chromatin and DNA Templates.

Negatively supercoiled pLY10 DNA template (9) was purified as described in ref. 47. The linear forms of pLY10 and pAN6 plasmids were purified after digestion with EagI or SspI restriction enzymes, respectively. In vitro reconstitution of chromatin on supercoiled pLY10 template was conducted by using various amounts (specified in Supporting Text, which is published as supporting information on the PNAS web site) of purified chicken erythrocyte core histones H2A/H2B and H3/H4 by step dialysis from 2 M NaCl (50).

Topological Analysis of Supercoiled DNA.

To obtain preparations of the pLY10 plasmid that are negatively supercoiled to various extents, the supercoiled plasmid (10 μg/ml) was incubated in the presence of chicken blood extract (0.1 μl of the extract per 1 μg of DNA) in 200 mM NaCl, 20 mM Tris·HCl (pH 8.0), 0.25 mM EDTA, and 5% glycerol for 1.5 h at 37°C in the presence of the following concentrations of chloroquine: 0 μg per ml (σ = −0.010 in the transcription buffer), 50 μg per ml (σ = −0.020), 100 μg per ml (σ = −0.034), 200 μg per ml (σ = −0.042), 300 μg per ml (σ = −0.052), 500 μg per ml (σ = −0.060), and 700 μg per ml (σ = −0.065). Relaxed pLY10 (σ = 0 in the transcription buffer) was obtained by linearization and religation in the transcription buffer. The relaxed and supercoiled plasmids were purified by extraction with phenol/chloroform followed by ethanol precipitation, and analyzed by electrophoresis in 0.6% agarose gel containing indicated concentrations of chloroquine.

Analysis of Unconstrained DNA Supercoiling Density (σ) on Chromatin Templates (Fig. 1).

The densities of unconstrained internucleosomal linker DNA supercoiling in chromatin (σ) were calculated as described in ref. 51. Total number of linker DNA helical turns (X) and total number of unconstrained DNA supercoils (Y) were calculated by using the values of σ determined for the original DNA preparations (Fig. 1) and for the relaxed chromatin samples (Figs. 1 and 6), and assuming that each nucleosome core occupies 150 bp of DNA (34). The linking number difference (σ) is equal to Y/X and has the corresponding (negative) sign.

In Vitro Transcription.

Conditions for in vitro transcription were optimized for maximal utilization of the pLY10 templates. Transcription was conducted as described in ref. 47. In all experiments where different templates were compared, identical amounts of the templates (DNA) were used.