Mapping in Vivo Chromatin Interactions in Yeast Suggests an Extended Chromatin Fiber with Regional Variation in Compaction
2008; Elsevier BV; Volume: 283; Issue: 50 Linguagem: Inglês
10.1074/jbc.m806479200
ISSN1083-351X
Autores Tópico(s)Chromosomal and Genetic Variations
ResumoThe higher order arrangement of nucleosomes and the level of compaction of the chromatin fiber play important roles in the control of gene expression and other genomic activities. Analysis of chromatin in vitro has suggested that under near physiological conditions chromatin fibers can become highly compact and that the level of compaction can be modulated by histone modifications. However, less is known about the organization of chromatin fibers in living cells. Here, we combine chromosome conformation capture (3C) data with distance measurements and polymer modeling to determine the in vivo mass density of a transcriptionally active 95-kb GC-rich domain on chromosome III of the yeast Saccharomyces cerevisiae. In contrast to previous reports, we find that yeast does not form a compact fiber but that chromatin is extended with a mass per unit length that is consistent with a rather loose arrangement of nucleosomes. Analysis of 3C data from a neighboring AT-rich chromosomal domain indicates that chromatin in this domain is more compact, but that mass density is still well below that of a canonical 30 nm fiber. Our approach should be widely applicable to scale 3C data to real spatial dimensions, which will facilitate the quantification of the effects of chromatin modifications and transcription on chromatin fiber organization. The higher order arrangement of nucleosomes and the level of compaction of the chromatin fiber play important roles in the control of gene expression and other genomic activities. Analysis of chromatin in vitro has suggested that under near physiological conditions chromatin fibers can become highly compact and that the level of compaction can be modulated by histone modifications. However, less is known about the organization of chromatin fibers in living cells. Here, we combine chromosome conformation capture (3C) data with distance measurements and polymer modeling to determine the in vivo mass density of a transcriptionally active 95-kb GC-rich domain on chromosome III of the yeast Saccharomyces cerevisiae. In contrast to previous reports, we find that yeast does not form a compact fiber but that chromatin is extended with a mass per unit length that is consistent with a rather loose arrangement of nucleosomes. Analysis of 3C data from a neighboring AT-rich chromosomal domain indicates that chromatin in this domain is more compact, but that mass density is still well below that of a canonical 30 nm fiber. Our approach should be widely applicable to scale 3C data to real spatial dimensions, which will facilitate the quantification of the effects of chromatin modifications and transcription on chromatin fiber organization. The higher order organization of chromatin is thought to play critical roles in processes such as gene expression. Although the structure of nucleosomes is known in detail, much less is known about higher levels of chromatin organization. The first level of organization beyond the nucleosome may involve formation of a 30-nm-thick chromatin fiber, but the precise organization of DNA within this structure and the range of compaction levels of this fiber are poorly understood (for reviews see, e.g. Refs. 1.Belmont A.S. Dietzel S. Nye A.C. Strukov Y.G. Tumbar T. Curr. Opin. Cell Biol. 1999; 11: 307-311Crossref PubMed Scopus (115) Google Scholar, 2.Widom J. Annu. Rev. Biophys. Biophys. Chem. 1989; 18: 365-395Crossref PubMed Scopus (145) Google Scholar, 3.van Holde K.E. Chromatin. Springer, Heidelberg1989Crossref Google Scholar, 4.van Holde K. Zlatanova J. J. Biol. Chem. 1995; 270: 8373-8376Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 5.Woodcock C.L. Dimitrov S. Curr. Opin. Genet. Dev. 2001; 11: 130-135Crossref PubMed Scopus (220) Google Scholar, 6.Hansen J.C. Annu. Rev. Biophys. 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At very low salt concentrations chromatin does not form 30-nm-thick fibers, and nucleosomes appear in a zigzag arrangement to form fibers with a low mass density of around 1–2 nucleosomes per 11 nm fiber (6.Hansen J.C. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 361-392Crossref PubMed Scopus (417) Google Scholar, 12.Thoma F. Koller T. Klug A. J. Cell Biol. 1979; 83: 403-427Crossref PubMed Scopus (1174) Google Scholar, 15.Bednar J. Horowitz R.A. Grigoryev S.A. Carruthers L.M. Hansen J.C. Koster A.J. Woodcock C.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14173-14178Crossref PubMed Scopus (442) Google Scholar, 21.Gerchman S.E. Ramakrishnan V. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7802-7806Crossref PubMed Scopus (112) Google Scholar). Under conditions that are thought to mimic the in vivo milieu, i.e. 150 mm salt and 1–5 mm Mg2+, chromatin becomes compact to form more typical 30 nm fibers with a mass density of 6 nucleosomes per 11 nm (15.Bednar J. Horowitz R.A. Grigoryev S.A. 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Mol. Biol. 2008; 381: 816-825Crossref PubMed Scopus (230) Google Scholar).Combined these observations suggest that in vivo the level of compaction of chromatin is not uniform across the genome, but is modulated by local differences in chromatin modifications, linker length, as well as by binding of additional non-nucleosomal factors. Domain-wide differences in compaction have been observed, and these differences were correlated with regional variation in gene density, independent of gene expression level (29.Gilbert N. Boyle S. Fiegler H. Woodfine K. Carter N.P. Bickmore W.A. Cell. 2004; 118: 555-566Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). More localized fluctuations in chromatin compaction have also been observed, e.g. at and around active promoters (30.Sabo P.J. Hawrylycz M. Wallace J.C. Humbert R. Yu M. Shafer A. Kawamoto J. Hall R. Mack J. Dorschner M.O. McArthur M. Stamatoyannopoulos J.A. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16837-16842Crossref PubMed Scopus (115) Google Scholar, 31.Gheldof N. Tabuchi T.M. Dekker J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 12463-12468Crossref PubMed Scopus (46) Google Scholar).Here we present a strategy that can be used to measure the average level of compaction of specific genomic segments in intact cells. In this approach, we combine spatial distance information obtained by in vivo observations, with information about the conformation of chromatin obtained by chromosome conformation capture (3C) 2The abbreviations used are: 3C, chromosome conformation capture; MES, 4-morpholineethanesulfonic acid; GFP, green fluorescent protein.2The abbreviations used are: 3C, chromosome conformation capture; MES, 4-morpholineethanesulfonic acid; GFP, green fluorescent protein. (32.Dekker J. Rippe K. Dekker M. Kleckner N. Science. 2002; 295: 1306-1311Crossref PubMed Scopus (2460) Google Scholar). We have applied this approach to a 95-kb GC-rich chromosomal domain on chromosome III in the yeast Saccharomyces cerevisiae. Our results show that this chromatin domain is highly flexible with a mass density of ∼1.2–2.4 nucleosomes per 11 nm, which is well below the value of 6 nucleosomes per 11 nm predicted for a canonical 30 nm fiber. A neighboring AT-rich domain was found to be more compact with a mass density between 1.8 and 3.6 nucleosomes per 11 nm. Our results are consistent with a highly extended chromatin fiber, with variation in compaction at the level of sub-chromosomal domains.EXPERIMENTAL PROCEDURESChromosome Conformation Capture—3C was performed with intact cells (strain NKY2997: MATa, ho::LYS2, lys2, ura3, nuc1::LEU2) as described for nuclei (32.Dekker J. Rippe K. Dekker M. Kleckner N. Science. 2002; 295: 1306-1311Crossref PubMed Scopus (2460) Google Scholar), with the following adaptations. Cells were grown in YPD medium (1% (w/v) Bacto-yeast extract, 2% (w/v) Bacto-peptone, 2% (w/v) glucose) until A600 = 1.0. Cells were resuspended in 200 mm Tris-Cl, pH 7.5, 1 mm dithiothreitol and incubated at room temperature for 2 min. Cells were resuspended in 0.4 m Sorbitol, 0.4 m KCl, 0.5 mm MgCl2, 40 mm sodium phosphate (pH 7.2) and incubated at 30 °C in the presence of 0.2 mg/ml Zymolyase for 30 min. The resulting spheroplasts were washed three times in 0.1 m MES, 1.2 m Sorbitol, 1 mm EDTA, 0.5 mm MgCl2 (pH 6.4) and resuspended in the same buffer. Formaldehyde was added to a final concentration of 1%, and samples were incubated at room temperature for 10 min. Glycine was added to a final concentration of 125 mm, and samples were incubated for 5 min at room temperature. Chromatin was solubilized by adding SDS (0.1% final concentration) followed by incubation at 65 °C for 10 min. Triton was added to a concentration of 1%. Chromatin was digested with EcoRI at 37 °C for 18 h. Digestion efficiency was ∼70% and did not vary significantly between sites (Ref. 33.Dekker J. Genome Biol. 2007; 8: R116Crossref PubMed Scopus (46) Google Scholar, and not shown). EcoRI was inactivated by addition of SDS to a final concentration of 1.6%, and samples were incubated at 65 °C for 20 min. Chromatin was diluted to 2.5 ng/μl, and Triton X-100 was added to 1%. DNA was ligated for 2 h at 16 °C using T4 ligase. The cross-links were reversed by overnight incubation at 65 °C in the presence of 5 μg/ml Proteinase K, and DNA was purified by phenol-chloroform extraction and ethanol precipitation.A randomized control PCR template was generated by digestion and random ligation of purified yeast genomic DNA (NKY2997). The randomized control template contains all ligation products in equal molar ratios and is used to correct for differences in efficiency of PCR amplification of 3C ligation products. All templates were titrated to determine the linear range of PCR, and all quantifications of interaction frequencies were performed using template concentrations that were in the linear dynamic range of PCR. All primers were designed in a unidirectional fashion along the yeast genome to prevent detection of self-ligated partial digestion products (see Refs. 32.Dekker J. Rippe K. Dekker M. Kleckner N. Science. 2002; 295: 1306-1311Crossref PubMed Scopus (2460) Google Scholar, 34.Miele A. Gheldof N. Tabuchi T.M. Dostie J. Dekker J. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, NJ2006: 21.11.21-21.11–20Google Scholar).PCR products were quantified on 1.5% agarose gels in the presence of ethidium bromide. Interaction frequencies between loci were calculated in triplicate by determination of the ratio of the amount of ligation product detected by PCR with the 3C template divided by the amount of ligation product detected by PCR with the randomized control template (32.Dekker J. Rippe K. Dekker M. Kleckner N. Science. 2002; 295: 1306-1311Crossref PubMed Scopus (2460) Google Scholar).Determination of Cross-linking Efficiency—We used data obtained by Nagy and co-workers (35.Nagy P.L. Cleary M.L. Brown P.O. Lieb J.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6364-6369Crossref PubMed Scopus (87) Google Scholar) to determine the level of formaldehyde induced cross-linking throughout the AT- and GC-rich domains of chromosome III. Using data from their experiment dl_g_067K Exp. #28 ("ORF-enrichment analysis," see Ref. 35.Nagy P.L. Cleary M.L. Brown P.O. Lieb J.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6364-6369Crossref PubMed Scopus (87) Google Scholar for details) we determined the enrichment of open reading frames in the cross-linked fraction. The average enrichment (Log2) for genes in the AT-rich domain (n = 37) was 0.04 (S.E. ± 0.057). The enrichment (Log2) for genes in the GC-rich domain (n = 59) was 0.25 (S.E. ± 0.042). The difference in enrichment (Log2) was 0.21 (S.E. ± 0.071). This corresponds to a 1.15-fold higher cross-linking efficiency for chromatin in the GC-rich domain. When we analyzed the enrichment of intergenic regions (n = 39 for the AT-rich domain, n = 46 for the GC-rich domain) in the cross-linked fraction we found a similar fold difference (1.13-fold p < 0.01, t test).RESULTSThe mass density of chromatin will affect the average distance between two loci. However, the spatial distance between two loci does not by itself provide sufficient information to determine the level of compaction. This is illustrated by two extreme situations in Fig. 1. The chromatin fiber can be very compact and relatively stiff (Fig. 1, A and B, left panels) or the fiber can be relatively extended and highly flexible (Fig. 1, A and B, right panels). In these examples differences in flexibility allow two loci to be separated by the same spatial distance despite significant differences in chromatin compaction. Therefore, the mass density of the chromatin fiber can only be determined when both the average distance between two loci and the flexibility of the intervening chromatin fiber are known (Fig. 2A).FIGURE 2Determination of mass density in intact yeast cells. A, schematic representation of the strategy to measure the level of chromatin compaction. The spatial distance between the HMR and MATa loci was determined by targeting GFP to these sites followed by fluorescence microscopy and three-dimensional reconstitution. The spatial conformation of the intervening chromatin was determined by 3C. B, spatial distances between HMR and MATa. Data are taken from Simon and co-workers (36.Simon P. Houston P. Broach J. EMBO J. 2002; 21: 2282-2291Crossref PubMed Scopus (16) Google Scholar). Distances were grouped in bins of 300 nm and the frequency of each bin is plotted. The average spatial distance was determined as described in the text. C, interaction frequencies between a number of loci located between HMR and MATa were determined in triplicate by 3C and plotted against site separation (see Table 2). Error bars indicate standard error of the mean. The arrow indicates the data point corresponding to the interaction between HMR and MATa. The solid line indicates the fit to Equation 4. D, Mass density L was plotted against three-dimensional spatial distance between HMR and MATa (r(HMR-MATa)) using Equation 9 for X(HMR-MATa) = 0.38 and [k × L–3] = 1002 m–1 nm–3 kb3 (solid line) and for [k × L–3] = 1460 m–1 nm–3 and 544 m–1 nm–3 (dotted lines; these values represent the 95% confidence interval based on the standard deviation of [k × L–3]). The range of values for L are indicated that correspond to r(HMR-MATa) = 463 nm ± two times the standard deviation (33 nm).View Large Image Figure ViewerDownload Hi-res image Download (PPT)We describe a strategy that combines in vivo distance data with measurements of chromatin flexibility in intact cells to determine the mass density of chromatin and applied it to analysis of a 95-kb GC-rich chromatin domain on the right arm of chromosome III of the yeast S. cerevisiae (Fig. 2A). This domain is flanked by the MATa locus on the left and the HMR locus on the right.Determination of the Average Spatial Distance between MAT and HMR—The spatial distance between MATa and HMR has been analyzed in a cells (36.Simon P. Houston P. Broach J. EMBO J. 2002; 21: 2282-2291Crossref PubMed Scopus (16) Google Scholar). These authors introduced Lac repressor binding sites at the MATa locus and Tet repressor binding sites at the HMR locus in a strain expressing GFP-LacI and GFP-TetR fusion proteins. Binding of these fusion proteins to their binding sites resulted in the formation of fluorescent spots that marked the positions of HMR and MATa in living cells (see Ref. 36.Simon P. Houston P. Broach J. EMBO J. 2002; 21: 2282-2291Crossref PubMed Scopus (16) Google Scholar for details). Fluorescence microscopy and three-dimensional reconstruction were used to determine the position of each locus. We used the data from Simon et al. (36.Simon P. Houston P. Broach J. EMBO J. 2002; 21: 2282-2291Crossref PubMed Scopus (16) Google Scholar) to determine the average spatial distance between HMR and MATa. In this dataset distances smaller than 200 nm could not be resolved as the two GFP spots merged into one (36.Simon P. Houston P. Broach J. EMBO J. 2002; 21: 2282-2291Crossref PubMed Scopus (16) Google Scholar). For cells in which only a single GFP spot was observed, the spatial distance was therefore between 0 and 200 nm, and we assumed the average spatial distance in this sub-population to be 100 nm. For the remaining cells where two GFP spots could be detected, the actual measured three-dimensional distance was used (Fig. 2B). We determined that the average spatial distance between HMR and MATa for the entire cell population was 463 nm (S.E. = 33 nm). This distance is comparable to data reported by Bystricky and co-workers (37.Bystricky K. Heun P. Gehlen L. Langowski J. Gasser S.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16495-16500Crossref PubMed Scopus (223) Google Scholar) who found that pairs of loci on yeast chromosome 14 separated by 90 kb were located on average 440 nm apart, and a pair of loci on the same chromosome separated by 98 kb were 417 nm apart.Determination of Chromosome Conformation of the MATa-HMR Domain—The conformation and flexibility of chromatin in intact cells can be determined using the 3C method (32.Dekker J. Rippe K. Dekker M. Kleckner N. Science. 2002; 295: 1306-1311Crossref PubMed Scopus (2460) Google Scholar). 3C is used to determine interaction frequencies between genomic loci using formaldehyde cross-linking followed by detection and quantification of cross-linked sequences by PCR. 3C has been described in detail elsewhere (32.Dekker J. Rippe K. Dekker M. Kleckner N. Science. 2002; 295: 1306-1311Crossref PubMed Scopus (2460) Google Scholar, 33.Dekker J. Genome Biol. 2007; 8: R116Crossref PubMed Scopus (46) Google Scholar, 38.Dekker J. Trends Biochem. 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A. 1995; 92: 2710-2714Crossref PubMed Scopus (249) Google Scholar, 44.Ostashevsky J.Y. Lange C.S. J. Biomol. Struct. Dyn. 1994; 11: 813-820Crossref PubMed Scopus (32) Google Scholar, 45.Rippe K. von Hippel P.H. Langowski J. Trends Biochem. Sci. 1995; 20: 500-506Abstract Full Text PDF PubMed Scopus (251) Google Scholar, 46.Ringrose L. Chabanis S. Angrand P.O. Woodroofe C. Stewart A.F. EMBO J. 1999; 18: 6630-6641Crossref PubMed Scopus (139) Google Scholar, 47.Rippe K. Trends Biochem. Sci. 2001; 26: 733-740Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar).We used 3C to determine the conformation and flexibility of the chromatin fiber between HMR and MATa. Previously we have analyzed this chromosomal region using 3C with nuclei isolated from α-factor-arrested cells. Because the three-dimensional distance data were obtained using non-arrested intact cells, we wished to perform 3C using intact yeast cells as well, which required minor adaptations of our 3C protocol (see Refs. 32.Dekker J. Rippe K. Dekker M. Kleckner N. Science. 2002; 295: 1306-1311Crossref PubMed Scopus (2460) Google Scholar, 34.Miele A. Gheldof N. Tabuchi T.M. Dostie J. Dekker J. Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley & Sons, Hoboken, NJ2006: 21.11.21-21.11–20Google Scholar and "Experimental Procedures" above). Using intact cells we determined 30 interaction frequencies between 19 restriction fragments located in between MATa and HMR (see Table 1 for 3C primer sequences and Table 2 for 3C data). Fig. 2C shows the interaction frequencies plotted against the site separation of the interacting loci (in kilobases).TABLE 13C primer sequencesPrimer numberPrimer sequence82GGTGGCACTTCTTCCTCTTGTTCTTATAG83AGCCATTCGCCGGTTTGGTTGACGCTAA84ATCCTTGCTTTACAATCACCGCTACCAG85CTGTCTTTAAATTGAGAGTTTCCAGAAAGG123CTTCCTCTTCATCGTGCTCAGGCGTATC124CCGGTAGAATTTCCCCAATTCAAGAAGAAC125GCCGAAGATCAATTTTACGTCTATGACTCA126ACGTACTTCCTCTTTGAGCTAAAGGCGTTA127ACGTTGCATGATGTGGCTGCTAACGACA145GAGACTCCAATCAGGTATATCACGACGC146CGCTTTCTGATTGTTCTACTATTAACGAGG147CAAGAACCCCTATGGACTACTAATGCCG148CCCAGATGTGGGTCCTTTCACCGTCTAT149GGGCACACCATCAAAATCCTCTTGGCAA150GTCTGCCAGCAACGCAAATGACTTTCAAT151CCCTCTTTCAGCTATATTGTTCAAGGAGTA152GTACTCACTGAACAGGAAATTCTCGCGACA182CACCGCTACCAGTAGCAAAAACGTTATATTCTG273GGCCACCCTCCATGTTAGACCACCGTCT79CGCCAATCCAGCAATACCTGTACCTCTA80CTCGTTATTAGTAGGTCGTGCTCTTAAAAG81TGGTTTGGCCATCTCTTCTTGGTTGCGA82GGTGGCACTTCTTCCTCTTGTTCTTATAG119AATCATTAATGGTGAGAGCAAATCCTCTACC120GGTATTCACGGTCATCGGAATCTCAGTTC121GCACAATTCAACTTGAGCACGCACACTAAG135TTCTGAAAACCGCTGATATCATTGGAGACG136GAAGGTATTCCAGCTGGCTGGCAAGGGT137GGTTGATCATTCTGTCTTCAGATGCCCAC138TGCAGTTGGCGACGTATTGGTGGAGACT139CGTTCGGTACATAACCGAGAATTAACTGTG140GGTGAGATTCAAATCTCCAGACCGGATG143CCATTGCTGAAAATCGCAAAACCCACAGAG174GTGGAGACGCAGTTCTTGAGCCGTCAGG Open table in a new tab TABLE 23C interaction frequencies (arbitrary units)Primer 1Primer 2Site separationInteraction frequencyS.E.SSkbGC-rich domain 8314571.150.061 82123101.250.070 83125110.960.020 145148111.350.059 148150120.840.070 83146130.650.028 83147150.720.049 83148180.420.041 125150190.680.050 83126210.590.052 82124220.900.036 83149240.400.040 148151280.660.054 83150300.420.052 8283300.390.033 83182370.340.028 150273390.460.050 146127430.500.036 14885460.410.055 82126510.300.025 273147540.340.011 12384570.430.022 82150600.190.009 124152620.380.026 27383690.300.010 123152740.370.062 123127750.270.033 82152840.240.044 82127860.180.024 123273950.310.014AT-rich domain 1218170.940.115 13511991.540.311 81140101.510.237 139140150.710.068 121140170.840.065 12081181.340.040 135120181.090.078 138140221.050.029 119121280.720.095 8182280.500.071 13781300.590.026 136121300.560.053 136139320.240.019 135121350.520.032 13681370.430.183 135139370.270.048 13581420.540.014 119140450.480.022 136140470.380.028 135140520.460.036 119143580.390.059 136143630.340.016 174140660.070.012 135143660.370.029 13582700.100.012 8082780.160.045 79143850.100.032 Open table in a new tab Next we fitted the 3C data to a polymer model to determine chromatin flexibility. Previously we successfully used a model that combines the Kratky-Porod worm-like chain model and the freely jointed chain model to describe cross-linking frequencies of loci along chromatin fibers in isolated nuclei (32.Dekker J. Rippe K. Dekker M. Kleckner N. Science. 2002; 295: 1306-1311Crossref PubMed Scopus (2460) Google Scholar, 46.Ringrose L. Chabanis S. Angrand P.O. Woodroofe C. Stewart A.F. EMBO J. 1999; 18: 6630-6641Crossref PubMed Scopus (139) Google Scholar, 47.Rippe K. Trends Biochem. Sci. 2001; 26: 733-740Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). The facts that the region we analyze here appears relatively unconstrained in its movement (48.Bressan D.A. Vazquez J. Haber J.E. J. Cell Biol. 2004; 164: 361-371Crossref PubMed Scopus (50) Google Scholar) and that end-to-end distances appear to display a Gaussian distribution (36.Simon P. Houston P. Broach J. EMBO J. 2002; 21: 2282-2291Crossref PubMed Scopus (16) Google Scholar, 37.Bystricky K. Heun P. Gehlen L. Langowski J. Gasser S.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16495-16500Crossref PubMed Scopus (223) Google Scholar) support the use of a polymer model to describe the domain.The model is given in Equations 1 and 2 and describes the relationsh
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