Cell-to-Cell Diversity of Nucleosome Positioning.

As a model for determining cell-to-cell diversity of nucleosome positioning, we used the PHO5 promoter as the position of its nucleosomes was well characterized in bulk studies (canonical nucleosome positioning depicted in Fig. 1A). PCR-based nucleosome-scanning assay (Fig. 1B) and genome-wide micrococcal nuclease (MNase-seq) were performed (Fig. 1C), confirming previously reported nucleosome positioning on the PHO5 promoter for positions of the +1 through −3 nucleosomes of the PHO5 promoter under phosphate-rich conditions, which is nonpermissive for gene expression (30, 32⇓⇓–35).

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Fig. 1.

Nucleosome architecture of the PHO5 promoter can be ascertained in individual cells and demonstrates heterogeneity. (A) Canonical position of nucleosomes (gray ovals) in the PHO5 promoter. White circles indicate the locations of cytosines that are part of GC dinucleotides. (B) Mapping of mononucleosomal DNA of cells grown in rich media using a nucleosome-scanning assay. Chromatin was digested with MNase, mononucleosomal DNA was purified, and MNase protection was determined by quantitative PCR. Enrichment of mononucleosomal DNA (y axis) is indicated by the midpoints of each amplicon (x axis), and error bars represent 1 SD from two independent biological replicates. (C) MNase-seq track of the PHO5 promoter from cells grown in rich media. (D) Nucleosome architecture of 806 cells from three bulk populations revealed eight conformations (a to h). Nucleosomes are depicted as gray ovals. Red circles indicate methylated cytosines, and white circles indicate unmethylated cytosines that are part of GC dinucleotides. The fraction of total cells that demonstrated each protection pattern is indicated on the right. The SDs for the three experiments were all less than 1.5%.

To map nucleosomes in individual cells, we modified a method developed in the Kladde and Jones laboratories based upon the ability of nucleosomes to block the GpC-specific DNA methyltransferase M.CviPI from modifying DNA wrapped around a histone octamer (34, 36, 37). In comparison with other nucleosome-mapping methods, this method is less destructive than MNase for mapping the DNA at the ends of the nucleosome or chemical mapping, which cleaves the DNA at the center of the nucleosome (19). MNase cleavage also has sequence specificity, which can bias the results of nucleosome mapping (38⇓⇓–41). Although chemical mapping nucleosomes by incorporation of a modified histone circumvents the sequence specificity, it is not a very efficient reaction and thus can only map a fraction of the nucleosomes within a cell (19, 41).

To map nucleosomes in single cells, yeast are converted to permeable spheroplasts and treated with M.CviPI, which methylates the cytosines of GpC dinucleotide that are not protected by DNA-bound proteins including nucleosomes. Single cells were isolated by serial dilution until there was, on average, zero or one cell per well. DNA from single cells was extracted, digested, and treated with sodium bisulfite to convert unmethylated cytosines to uracil. Each cellular equivalent of DNA was subjected to PCR amplification using primers complimentary to the promoter of interest, and DNA amplicons were sequenced. The pattern of GpC methylation within the promoter of each single cell was compared with all possible GpC sites in the promoter to infer the locations of nucleosomes (Fig. 1D).

It is important to note that this method allows for the inference of nucleosome location by protection patterns of the GpCs and therefore does not reveal information regarding nucleosomes being smaller or larger than 147 bp. Moreover, the methyltransferase activity and the bisulfite conversion needed to be as close to 100% efficient as possible to accurately map nucleosomes in individual cells. If any cytosine that should have been methylated or bisulfite-converted were not modified, this would give an inaccurate result. To measure enzymatic efficiency, a time course was performed with M.CviPI followed by bisulfite sequencing to determine the incubation time necessary to achieve methylation of all accessible GpCs (Fig. S1).

In our pilot study of 235 cells, 212 did not have a nucleosome over the low-affinity upstream activating sequence (UAS), a pattern further supported in our final data, explained in detail below (Fig. 1D, conformations a to f). Of the pilot set, only six cells had unprotected GpCs flanking the low-affinity UAS, suggesting transcription factor binding. Two other cells had a single unprotected GpC among the seven GpCs between the −2 and −3 nucleosomes. These single unprotected GpCs could be attributable to an inability of the methyltransferase to access this region of DNA because of a protein factor or secondary nucleic acid structure or inefficient methyltransferase or bisulfite conversion (i.e., less than 100% efficiency); however, taken together, the data from the pilot experiment strongly indicated that methylation and bisulfite conversion are extremely effective and thus can be used for single-cell nucleosome mapping.

We further note that as a quality control for each single-cell nucleosome-mapping experiment, bulk genomic DNA was treated with M.CviPI followed by bisulfite conversion and cloning; 25 colonies were sequenced to confirm that all cytosines within GpCs were methylated and all free cytosines were bisulfite-converted. Moreover, to corroborate that PCR products of single-cell experiments indeed represented DNA from a single cell, several PCR products from a single cell were cloned from each experiment and a minimum of 10 colonies were sequenced and shown to be exactly the same. Together, these controls demonstrate that GpC methyltransferase activity and bisulfite conversion are ∼99% efficient and PCR amplicons do represent single cells. Thus, we concluded that the technique described above could be used to determine GpC protection patterns of individual cells.

Using single-cell mapping, we examined nucleosome occupancy in three biological replicate mapping experiments, including the pilot examination of 235 cells, for a total of 806 single cells (Fig. 1D). We observed that in a population of cells grown under nutrient-rich, nonpermissive conditions for PHO5, the majority of individual cells demonstrated nucleosome architecture that resembles the canonical nucleosome map (compare Fig. 1 A and D). Specifically, 32% of cells had promoters in the same nucleosome configuration that fits the canonical nucleosome map (Fig. 1D, conformation a). An additional 11% of the cells demonstrate only a slight difference of one GpC at the 3′ end of the −1 nucleosome (compare conformations a and c in Fig. 1D), which may be attributable to a small shift in the nucleosome location. It is also possible that there is a difference in the ability of the methyltransferase to modify that particular GpC because there is a single base that separates the two GpCs. Approximately 91% of all cells had a nucleosome-rich promoter with nucleosomes occluding both the TATA box and high-affinity UAS and a nucleosome-free low-affinity UAS (Fig. 1D, conformations a to f). These data suggest that generally only small variations in nucleosome positions exist in the population.

Under nutrient-rich conditions, the PHO5 promoter displayed four well-positioned nucleosomes (Fig. 1D). The +1 nucleosome appears to shift only slightly, if at all, in any of the cells, which is in agreement with previous reports indicating that nucleosome positioning is strongest at the +1 position (3, 42). The −1 nucleosome that occludes the TATA box appears less well positioned by MNase-seq (Fig. 1C); however, upon closer investigation, there are still about 500 DNA sequence reads that span the 162-bp region (Fig. 1C), which is in agreement with the nucleosome-scanning assay (Fig. 1B).

The single-cell data further reveal more differences in positioning of the −1 nucleosome compared with the +1 nucleosome because four configurations (including the absence of the −1 nucleosome) were identified. The vast majority of the cells (Fig. 1D, conformations a to f) demonstrated nucleosome occupancy at the −1 position in two different protection patterns: ∼6% of the cells had a nucleosome shifted away from the TATA box, and only ∼4% of the cells did not show the presence of a nucleosome at the −1 position. The −2 nucleosome was also well positioned with only small cell-to-cell variations. The −2 nucleosome was easier to map accurately compared with the −1 based on GCs located on each end of the nucleosome, and tight positioning correlated with the relatively sharp peak in the MNase-seq data (Fig. 1C). The majority of these cells had a −2 nucleosome occluding the high-affinity UAS, as previously shown by other methods (30, 32⇓⇓–35). However, the single-cell analysis showed that about 10% of cells do not have a nucleosome over the high-affinity UAS (Fig. 1D, conformations g and h) but instead have a nucleosome shifted 3′ to the −2 nucleosome that occludes the low-affinity UAS, a result that could not be ascertained from the bulk MNase-seq or nucleosome-scanning methods.

The −3 nucleosome demonstrated the most variation in positioning, which is in agreement with the MNase-seq track and nucleosome-scanning assay. The two peaks in the MNase-seq are in very close proximity in a region too small to accommodate two nucleosomes, so the exact position of these nucleosomes is more difficult to ascertain from bulk methods. The single-cell data indicated that the majority of cells occupy one of two conformations at the −3 position (Fig. 1D, confirmations a to e), but almost 20% of the population has a nucleosome that spans the region between the two conformations (Fig. 1D, confirmations f to h). Taken together with the data above, this single-cell analysis illustrates that although many cells conform to the general pattern of the nucleosome architecture defined by assay of a bulk population, at the single-cell level, considerable variability of nucleosome positioning exists.

Single-Cell Mapping Deconvolutes Fuzzy Nucleosomes.

Although the PHO5 promoter was chosen as one model because of its well-positioned nucleosomes, our single-cell analysis revealed distinct cell-to-cell variation in exact nucleosome positions. Nucleosome positioning can range from almost perfect positioning (e.g., the +1 nucleosome of PHO5), with no variability, to undefined positioning, where a nucleosome can be located at many sites within a region of DNA. MNase-seq and other nucleosome-mapping techniques can yield fuzzy nucleosomes in which a broad region without a peak demonstrates partial protection from digestion. Only single-cell analysis can determine whether this “fuzziness” is caused by the nucleosome forming along a continuous stretch of DNA at numerous positions or is attributable to a smaller set of cells that contain discrete positions assumed by the nucleosome.

We examined two loci: the 5′ end of CHA1 and the promoter of CYS3, both of which display poorly characterized nucleosomes by MNase-seq (Fig. 2). In the case of CHA1, the nucleosome-scanning assay and MNase-seq revealed two potential nucleosomes at the 5′ end of the gene followed by a fuzzy, or nucleosome-free, region depicted by a low amount of enrichment of mononucleosomal DNA in the nucleosome-scanning assay (Fig. 2A) and the broad flat region in the MNase-seq track (Fig. 2B). We analyzed a total of 481 cells and identified 68 different nucleosome conformations of four potential nucleosomes in a 575-bp region of CHA1. The seven most common conformations were seen in 31% of cells (Fig. 2C, conformations a to g). However, a great many more conformations exist, some of which were identified in only one cell (Fig. S2). It is highly likely that even more variability exists in this region because we are limited by our ability to only detect differences in positioning via GpC protection. Although nucleosome scanning and MNase-seq confirmed DNA sequences were protected from digestion, specific locations for nucleosomes are unclear. Single-cell analysis revealed that in the +3 and +4 nucleosome region, only 17% of cells were actually nucleosome-free and an additional 31% contained only one nucleosome in this region.

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Fig. 2.

Single-cell analysis of nucleosome architecture reveals distinct positions of nucleosomes previously described as fuzzy. (A) Nucleosome-scanning assay of the 5′ region of CHA1 from cells grown in rich media. The experiment was performed as described in Fig. 1. (B) MNase-seq track of CHA1 from cells grown in rich media. (C) Nucleosome architecture of 481 cells from two bulk experiments revealed 68 conformations of nucleosomes in the 5′ region of CHA1 (Fig. S2). An overlay of all possible nucleosome positions is depicted followed by the top seven conformations that represent 31% of the total population. (D) Nucleosome-scanning assay of the CYS3 promoter from cells grown in rich media. The experiment was performed as described in Fig. 1. (E) MNase-seq track of CYS3 promoter from cells grown in rich media. (F) Nucleosome architecture of 550 cells from two bulk experiments revealed 20 conformations of nucleosomes in the CYS3 promoter (Fig. S3). An overlay of all possible nucleosome positions is depicted, followed by the top six conformations that represent 31% of the total population.

Examination of the CYS3 promoter also revealed a collection of nucleosome conformations (Fig. 2F and Fig. S3). We analyzed 550 cells, and overall we found three nucleosomes positioned in 20 conformations spanning 540 bp in the promoter and 5′ end of CYS3. Nucleosome scanning and MNase-seq demonstrated a very well-defined nucleosome in the −2 and +1 positions of CYS3 (Fig. 2 D and E). We only detected one conformation for the nucleosome at the −2 position and four conformations at the +1 nucleosome that span a region of 166 bp (Fig. 2F and Fig. S3). By contrast, we detected five positions for the −1 nucleosome spanning a larger region of 184 bp, and in some cells, there was complete absence of a nucleosome in this region. In the majority of cases, a nucleosome is present in one of four conformations at the −1 position, but 36% of cells entirely lack a nucleosome in the promoter. These results demonstrate that our method can detect distinct nucleosome locations that previously were described as fuzzy or even nucleosome-free.

Nucleosome Remodeling Is Observed upon Phosphate Starvation.

Using our method for determining nucleosome positioning in single cells, we next directly tested the correlation between nucleosome positioning and gene expression through the study of the yeast PHO5 gene. When grown in nutrient-rich media, PHO5 expression is low to absent in yeast. Most of the yeast cells grown under these conditions displayed a nucleosome over the TATA box and high-affinity UAS (Fig. 1D, −1 and −2 nucleosomes), presumably blocking transcription factor accessibility and leading to gene silencing. Nevertheless, even under these nonpermissive conditions, about 10% of cells lacked a nucleosome over the high-affinity UAS site and the TATA box (Fig. 1D, conformations g and h).

PHO5 expression is induced upon phosphate starvation (ref. 31 and Fig. S4), and the nucleosomes of this gene were shown to undergo significant rearrangement upon phosphate starvation, as determined by electron microscopy (30) and methyltransferase accessibility (34). In agreement with previous studies (35, 43, 44), PCR-based nucleosome-scanning assay showed marked shifts in chromatin structure (Fig. 3A). The +1 nucleosome was positioned and present in the presence or absence of phosphate, but upon phosphate starvation, the −1 nucleosome was, on average, threefold less likely to be occupied, and the position of the −2 nucleosome was significantly shifted downstream (Fig. 3A). Indeed, previous predictions and experimental work suggested that the −2 nucleosome needed to be removed in order for the −1 nucleosome to remodel (30, 34). Single-cell nucleosome mapping showed that upon phosphate starvation and activation of the PHO5 gene, 76% of cells now had a nucleosome-depleted architecture, compared with only 10% when cells were grown in phosphate-rich media (Fig. 3B, conformations g and h). The +1 nucleosome was present in all cells regardless of nutrient status, but 50% of cells starved of phosphate lacked the −1 nucleosome (Fig. 3B, conformation h), and an additional 26% of the cells had the −1 nucleosome shifted away from the TATA box, allowing it to be accessible to protein factors (Fig. 3B, conformation g). Although these data confirmed patterns of nucleosome rearrangement upon phosphate starvation mapped in the bulk population of yeast, they also demonstrate that ∼25% of cells failed to undergo a remodeling of the nucleosome architecture and thus remained in a nucleosome-rich confirmation (Fig. 3B, conformations a to h), demonstrating a unique subpopulation of cells.

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Fig. 3.

Remodeling of nucleosomes upon phosphate starvation correlates with an increase in gene expression. (A) Nucleosome-scanning assay of the PHO5 promoter from cells grown in rich media (black) and shifted to media lacking phosphate (gray) for 3 h. The experiment was performed as described in Fig. 1. (B) The nucleosome architecture of cells grown in rich media (column 1 in the table to the right) is from the experiments in Fig. 1. Nucleosome architecture of 465 cells grown in rich media and shifted to media lacking phosphate (column 2, Phosphate starved) from three bulk experiments revealed the same eight conformations of nucleosomes for the PHO5 promoter, but there is a shift in a greater population of more nucleosome-free conformations (g and h). Nucleosome architecture of 638 cells expressing PHO5 from two bulk experiments (column 3, GFP cells). Cells that contain an EGFP tag on the C terminus of PHO5 were grown in rich media and sorted for GFP-positive cells, which represents ∼1% of the total population.

PHO5 expression is very low under phosphate-rich conditions. Theoretically, this level of expression could arise from low-level transcription from all cells or could result from gene silencing in most cells and transcription from a small minority of cells. A yeast strain in which the PHO5 gene contains a C-terminal EGFP tag was grown under nutrient-rich, nonpermissive conditions. Less than 1% of the cells were GFP-positive, which indicates that a low basal level of transcription was occurring at the PHO5 locus. These GFP-positive cells were sorted by FACS and processed for single-cell mapping. The sorted population showed the same eight conformations of nucleosomes that were observed in the unsorted population, but there was a significant shift to more cells displaying an open chromatin structure (Fig. 3B, conformations g and h, and Fig. S5). Specifically, 54% of the GFP-positive cells showed a loss of nucleosomes over the UAS and a loss or shift away of a nucleosome over the TATA box, compared with only 10% of cells in an unsorted population.

Furthermore, we found that when cells were grown in the permissive, low-phosphate conditions, GFP-negative cells demonstrated the same distribution of nucleosome architectures as GFP-negative cells grown in rich media (Fig. S5), specifically these cells had a strong tendency to have the −1 and −2 nucleosome sites occupied. These data strongly suggest that the small population of cells that remain in a relatively nucleosome-free configuration under nonpermissive conditions account for “leaky,” low-level basal gene expression. The shift from nucleosome-rich to nucleosome-free promoter configurations correlates directly with gene expression indicating that nucleosome remodeling is a barrier to regulate gene expression.

Altering DNA Sequence Shifts Nucleosome-Positioning Preferences.

DNA sequence itself plays a role in nucleosome positioning because nucleosome formation is affected by the overall ability for a 147-bp sequence to bend around the histone octamer (16). The homopolymeric sequence poly(dA:dT) is intrinsically stiff and strongly inhibitory to nucleosome formation (16, 21⇓–23). Poly(dA:dT) tracts of only 12–15 bp result in nucleosome-free regions of more than 150 bp (3, 45). Thus, we asked how the introduction of a poly(dA:dT) tract in the PHO5 promoter would affect nucleosome organization at the single-cell level.

We tested four mutations: two of which were tracts comprised of a mixture of As and Ts (PolyA_1 and PolyA_2), and two of which were poly(dA:dT) tracts (PolyA_3 and PolyA_4). These mutations all changed base composition but did not change the length of the promoter sequence and were not in regions believed to contain transcription factor binding sites. All but one of the mutations led to an increase in PHO5-GFP expression, as determined by flow cytometry (Fig. 4A). In the mixed A and T tract, PolyA_2 did not alter gene expression (Fig. 4A), nor did it significantly alter the nucleosome architecture, because the distribution of nucleosome locations was similar to that observed in wild-type cells (Fig. 4B, mutation 2). One possibility for the lack of change may be that the mixed A and T tract did not sufficiently stiffen the DNA to antagonize nucleosome formation. However, another mixed A and T tract, PolyA_1, demonstrated significant alterations in its nucleosome locations that were similar to the poly(dA:dT) tract mutants PolyA_3 and PolyA_4. These mutations all demonstrated the eight conformations previously seen in the wild type cells (Fig. 4B, conformations a to h), as well as two additional conformations that are nucleosome-free (Fig. 4B, conformations i and j).

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Fig. 4.

Changes in gene expression correlate with changes in nucleosome positioning. (A) PHO5 gene expression was determined from wild-type (WT) and PHO5 mutants containing a C-terminal EGFP tag. Gene expression was measured by the relative fluorescence intensity by flow cytometry. In the left graph, gene expression is shown for wild-type and polyA mutant cells grown in rich media. In the right graph, gene expression is shown for wild-type and periodicity mutant cells grown in rich media (black) or shifted to phosphate starvation media for three hours (gray). (B, Upper) The canonical nucleosome architecture of PHO5 as described in Fig. 1. The location of each of the polyA mutation is indicated by a number in a yellow star. (B, Lower) The ten nucleosome conformations (a to j) determined from analysis of WT and polyA mutants grown in rich media. Two bulk experiments were performed and the number of cells analyzed for each mutant was as follows: PolyA_1, 385 cells; PolyA_2, 412 cells; PolyA_3, 368 cells; and PolyA_4, 404 cells. The same eight conformations (a to h) were observed as in Fig. 1, along with two additional conformations (i and j). The table to the right shows each cell type and the percentage of cells that were determined to be in each conformation. Data for WT were shown in Fig. 1. (C, Upper) The canonical nucleosome architecture of PHO5 shown as described in Fig. 1. The location of each periodicity mutant is indicated by a number in a yellow star. (C, Lower) The eight nucleosome conformations (a to h) of WT and periodicity mutants are shown. Two bulk experiments were performed, and the number of cells analyzed for each mutant was as follows: Periodicity_1, 443 cells in rich media and 612 cells in phosphate starvation; and Periodicity_2, 461 cells in rich media and 602 cells in phosphate starvation. The table to the right shows each cell type under “Rich media” and “Phosphate starvation” conditions, and the percentage of cells that were determined to be in each conformation. In the table, the data shown for WT, Rich media is from Fig. 1 and WT, Phosphate starvation is from Fig. 3.

Whereas the wild-type PHO5 gene had a nucleosome in the −2 position in 90% of cells, poly A_1 contains a poly(dA:dT) tract located at the edge of the −2 nucleosome, which led to loss of the −2 nucleosome in 65% of cells. These cells were also missing a −1 nucleosome or the −1 nucleosome was shifted away from the TATA box (Fig. 4B, conformations g to j). Furthermore, introduction of a poly(dA:dT) tract in the −3 nucleosome (Fig. 4B, mutants 3 and 4) led to an increase in cells lacking both the −2 and −3 nucleosomes (Fig. 4B, configurations i and j), suggesting that changes in primary DNA sequence can alter the phasing of a set of nucleosomes. In the previously unidentified conformations i and j (Fig. 4B), the low- and high-affinity UAS sites and TATA box are devoid of nucleosomes. This change in nucleosome conformation strongly suggests that the introduction of the poly(dA:dT) tract directly antagonizes nucleosome formation and affects gene expression. It is notable, however, that the change of nucleosome positions is not uniform throughout the population of yeast cells. Despite the unfavorable thermodynamics of DNA bending, some cells harboring the poly(dA:dT) tracts maintained the canonical basal nucleosomal configuration of the PHO5 promoter (Fig. 4B, configuration a). These data indicate that although the DNA sequence of a promoter is one factor that plays a role in positioning nucleosomes, other factors such as the position of sequence-specific binding proteins play an important role. Single-cell analysis suggests that changes in DNA sequence alters the probability that a nucleosome may occupy a particular site and contributes to the potential of a gene to be activated or repressed.

Nucleosome formation is favored by the presence of stretches of dinucleotides AA/TT/TA on the face of the helical repeat that can directly interact with the histone (4, 19, 20). Nucleosome occupancy has been observed to increase dramatically when a DNA sequence is altered to fit 5-bp intervals of AA/TT/TA dinucleotides with GC dinucleotides (46). Therefore, we determined how altering the periodicity would change nucleosome positioning of the PHO5 promoter. We examined two mutations predicted to enhance the periodicity (Periodicity_1 and Periodicity_2). Under nutrient-rich conditions, these mutants exhibited a stabilized −3 nucleosome. Specifically, there was an increase in the proportion of cells in configurations c and d and decrease in configuration b in both mutants compared with wild type (Fig. 4C). Not surprisingly, neither mutation had an effect on gene expression when cells were grown under nutrient-rich conditions, because these mutations did not lead to changes in the −2 nucleosome, which occludes the high-affinity UAS.

Upon phosphate starvation, the periodicity mutants demonstrated decreased expression compared with the wild-type PHO5 gene (Fig. 4A, Right). We reasoned that these mutations stabilize the nucleosomes in a manner that antagonizes remodeling upon phosphate starvation. Single-cell mapping of nucleosomes of these mutants upon phosphate starvation revealed similar nucleosome positioning to that of the cells when grown in nutrient-rich media (Fig. 4C). Whereas 76% of cells lose nucleosomes in the −2 position and the −1 nucleosome shifts away from the TATA box under phosphate-starvation conditions in the wild-type cells (Fig. 4C, conformations g and h), the periodicity mutants demonstrated no increase in the number of cells in nucleosome-depleted conformations g and h. These data indicate that the mutations that enhance the AA/TT/TA periodicity stabilize the nucleosomes in a manner that antagonizes chromatin remodelers.