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. 2006 Nov 21;103(47):17690-5.
doi: 10.1073/pnas.0603819103. Epub 2006 Nov 13.

Chromatin structure can strongly facilitate enhancer action over a distance

Mikhail A Rubtsov  1 , Yury S Polikanov, Vladimir A Bondarenko, Yuh-Hwa Wang, Vasily M Studitsky
Affiliations

Chromatin structure can strongly facilitate enhancer action over a distance

Mikhail A Rubtsov et al. Proc Natl Acad Sci U S A. .

Abstract

Numerous DNA transactions in eukaryotic nuclei are regulated by elements (enhancers) that can directly interact with their targets over large regions of DNA organized into chromatin. The mechanisms allowing communication over a distance in chromatin are unknown. We have established an experimental system allowing quantitative analysis of the impact of chromatin structure on distant transcriptional regulation. Assembly of relaxed or linear DNA templates into subsaturated chromatin results in a strong increase of the efficiency of distant enhancer-promoter E-P communication and activation of transcription. The effect is directly proportional to the efficiency of chromatin assembly and cannot be explained only by DNA compaction. Transcription activation on chromatin templates is enhancer- and activator-dependent, and must be accompanied by direct E-P interaction and formation of a chromatin loop. Previously we have shown that DNA supercoiling can strongly facilitate E-P communication on histone-free DNA. The effects of chromatin assembly and DNA supercoiling on the communication are quantitatively similar, but the efficiency of enhancer action in subsaturated chromatin does not depend on the level of unconstrained DNA supercoiling. Thus chromatin structure per se can support highly efficient communication over a distance and functionally mimic the supercoiled state characteristic for prokaryotic DNA.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Presence of unconstrained negative DNA supercoiling does not affect enhancer-dependent, distantly activated transcription in chromatin. (A) Experimental approach (see Results for details). (B) Effect of chromatin assembly on the level of unconstrained DNA supercoiling (σ). Two preparations of the pLY10 template having initial linking number differences (σINIT) −0.055 (dashed line) or −0.065 (solid line) were assembled into chromatin (0%, 40%, or 75% histone loading), and the level of unconstrained DNA supercoiling (σ) was determined. The glnAp2 promoter and NtrC-dependent enhancer are positioned 2.5 kb from each other on the pLY10 plasmid. (C) Efficiencies of transcription of the chromatin templates. Chromatin samples having various levels of histone loading and unconstrained DNA supercoiling (see Fig. 1B) were transcribed. Quantitative analysis of the specific transcripts. Note that 75% chromatin preparations containing very different levels of unconstrained DNA supercoiling are transcribed with similar efficiencies.
Fig. 2.
Fig. 2.
Chromatin assembly results in strong activation of enhancer-dependent, distantly activated transcription. (A) Experimental approach. The templates were incubated with or without topoisomerase I before adding ATP. (B) Chromatin assembled on supercoiled DNA (σ = −0.055) at 0%, 40%, 75%, and 95% histone loading was transcribed before (S) or after (R) DNA relaxation with topoisomerase I analysis of labeled transcripts by denaturing PAGE. (C) Quantitative analysis of the specific transcripts formed on supercoiled (SC) and relaxed (REL) templates. Note that assembly of 75% chromatin results in strong activation of transcription of the relaxed template.
Fig. 3.
Fig. 3.
Assembly of linear DNA into chromatin results in strong increase of the rate of E–P communication over a large distance. (A) Experimental approach. The rate of formation of the open complexes [that reflects the rate of communication (9)] on linear (Lin.), supercoiled (−SC) 65% linear chromatin pLY10 templates was measured by using a single-round transcription assay. The approach is described in Fig. 1A, but ATP was added for 0, 0.5, 1, 2, 4, 8, 16, or 32 min to allow E–P communication for various time intervals. (B) Analysis of labeled transcripts by denaturing PAGE. ST, specific transcripts; LC, loading control. (C) Quantitative analysis of the specific transcripts. Note that assembly of 65% chromatin results in utilization of a smaller fraction of templates (≈22%), but the rate of E–P communication within the active fraction is much higher than on linear histone-free DNA.
Fig. 4.
Fig. 4.
Transcription of 65% linear chromatin requires presence of all critical components of the system. Negatively supercoiled (−SC) or linear (Lin.) pLY10 DNA, or 65% chromatin assembled on linear pLY10 (Lin. Chr.) were transcribed as described in the Fig. 1A. The reactions contained all components required for enhancer-dependent transcription, or one of the components was missing as indicated. Note that in all cases the specific transcripts are formed only when the reactions contain all critical components of the system. M, end-labeled pBR322-MspI digest.
Fig. 5.
Fig. 5.
Chromatin structure supports high DNA compaction and efficient communication between distantly positioned DNA regions. (A) Relaxed histone-free DNA is not a compact structure and does not support efficient communication between distantly positioned DNA regions. On supercoiled histone-free DNA, two double helices are in close proximity and efficient communication between any two DNA regions by the slithering mechanism is possible (9, 12), but this is not a compact structure. Chromatin structure can support both high compactness and efficient communication between distant DNA regions. (B) A “dynamic DNA unwrapping” model of chromatin organization. Because wrapping–unwrapping of nucleosomal DNA occurs at a high rate (39), chromatin not only compacts DNA and brings distant DNA regions closer to each other but also creates additional points of dynamic protein-induced DNA flexibility (at the points of entrance and exit of nucleosomal DNA). This may result in formation of both compact and flexible chromatin structure.
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