Mapping in vivo chromatin interactions in yeast suggests an extended chromatin fiber with regional variation in compaction

Abstract

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.

FIGURE 1.

The spatial distance between two loci is dependent on the conformation and compaction of the intervening chromatin. A, the left panel shows the path (in gray) of a compact and relatively stiff chromatin fiber. The right panel illustrates the path of an extended and more flexible fiber. Despite these differences two loci (open circles) can be separated by a similar three-dimensional spatial distance (dotted line). B, the left panel illustrates hypothetical packing of nucleosomes (gray circles) to form a compact fiber (11 nm/kb or 6 nucleosomes per 11 nm). The right panel depicts a hypothetical extended fiber that is composed of a three-dimensional zigzag of nucleosomes (33 nm/kb or 2 nucleosomes per 11 nm).

FIGURE 2.

Determination 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). 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).

FIGURE 3.

Regional variation in mass density. Interaction frequencies between a number of loci located within the AT-rich domain of chromosome III (position 100–190 kb) were determined in triplicate by 3C and plotted against site separation (see Table 2). Error bars indicate standard error of the mean. The solid line indicates the fit to Equation 4.

FIGURE 4.

Determination of mass density using the average spatial distance for loci separated by 46 kb. Mass density L was plotted against three-dimensional spatial distance using Equation 9. The solid line is for the pair of loci located in the GC-rich that are separated by 46 kb (X₍₄₆₎ = 0.41, see Table 2). The dotted lines are for two pairs of loci located in the AT-rich domain and separated by 45 kb (X₍₄₅₎ = 0.48) and 47 kb (X₍₄₇₎ = 0.38). We used [k × L^–3] = 1002 m^–1 nm^–3 kb³ for loci in the GC-rich domain (solid line) and [k × L^–3] = 3104 m^–1 nm^–3 for loci located in the AT-rich domain (dotted lines). The values for L are indicated that correspond to r₍₄₆₎ = 373 nm.