DNA-guided establishment of nucleosome patterns within coding regions of a eukaryotic genome

Abstract

A conserved hallmark of eukaryotic chromatin architecture is the distinctive array of well-positioned nucleosomes downstream from transcription start sites (TSS). Recent studies indicate that trans-acting factors establish this stereotypical array. Here, we present the first genome-wide in vitro and in vivo nucleosome maps for the ciliate Tetrahymena thermophila. In contrast with previous studies in yeast, we find that the stereotypical nucleosome array is preserved in the in vitro reconstituted map, which is governed only by the DNA sequence preferences of nucleosomes. Remarkably, this average in vitro pattern arises from the presence of subsets of nucleosomes, rather than the whole array, in individual Tetrahymena genes. Variation in GC content contributes to the positioning of these sequence-directed nucleosomes and affects codon usage and amino acid composition in genes. Given that the AT-rich Tetrahymena genome is intrinsically unfavorable for nucleosome formation, we propose that these "seed" nucleosomes--together with trans-acting factors--may facilitate the establishment of nucleosome arrays within genes in vivo, while minimizing changes to the underlying coding sequences.

Figure 1.

In vivo-like nucleosome organization without trans-acting factors. (A) Histograms of nucleosome positions relative to the TSS were computed from yeast and Tetrahymena MNase-seq data using the same bioinformatic pipeline. A phased distribution of nucleosome positions downstream from the TSS is observed in chromatin from log-phase Tetrahymena and yeast grown in rich media. Surprisingly, an in vivo-like pattern of nucleosome positioning is observed in vitro for Tetrahymena but not yeast. (B) Averaged nucleosome dyad counts around the TSS reveal an in vivo-like distribution of called nucleosomes within in vitro data. MNase-digested naked DNA does not resemble in vivo data (green curve), thus ruling out potential sequence biases associated with MNase preferences.

Figure 2.

Nucleosome organization in the Tetrahymena genome. (A) Stereotypical nucleosome organization near the 5′ end of genes in a large genomic region. Vertical black arrows represent the TSS, while light purple boxes represent 5′ UTRs. (B) The in vitro nucleosome organization at individual genes resembles in vivo patterns. Diagonal black lines indicate the presence of in vitro nucleosomes at in vivo-like locations. Most genes exhibit a subset of nucleosomes at standard positions in vitro.

Figure 3.

Distinct nucleosome patterns underlie similar aggregate patterns in Tetrahymena. (A) Individual genes were annotated as either possessing or lacking standard nucleosomes at the +1, +2, and +3 positions. For example, the pattern represented by a nucleosome only at the +1 position denotes genes with a +1, but not a +2 or +3 nucleosome. Standard nucleosomes in individual genes were annotated if they lie ≤35 bp from the aggregate position. The aggregate positions are +113, +306, +498, for log-phase (in vivo); +114, +307, +497 for starve; and +122, +310, +505 for in vitro, as defined from the peak positions in Figure 1B. The dominant pattern for in vivo data sets is the full nucleosome array, while a distribution of patterns is observed for in vitro data sets. (B) Standard nucleosome positions are preferred in vitro for Tetrahymena but not yeast. For each gene, the number of nucleosomes in standard and “nonstandard” positions was calculated. Then, the ratio of the total number of standard nucleosomes to nonstandard nucleosomes was calculated across all genes. Standard nucleosomes are defined as in A. Nonstandard nucleosomes lie ≤35 bp from the midpoint between the aggregate +1/+2 and +2/+3 positions, respectively. These are calculated as (113 + 306)/2 = 210 and (306 + 498)/2 = 402 for log-phase; (114 + 307)/2 = 211 and (307 + 497)/2 = 402 for starve; (122 + 310)/2 = 216 and (310 + 505)/2 = 408 for in vitro. As expected, the standard/nonstandard nucleosome ratio is high in vivo for both organisms. However, this ratio is threefold as high in vitro for Tetrahymena compared to yeast.

Figure 4.

Standard in vitro nucleosomes coincide with GC content oscillations and are associated with increased nucleosome positioning in vivo. Tetrahymena genes were classified according to the number of standard in vitro nucleosomes downstream from their TSS. Nucleosome positioning data are obtained from in vitro (blue line) and in vivo (red line) experiments, as well as from predictions of a thermodynamic model formulated by Kaplan et al. (2009) (black line). Log-phase MNase-seq data were used as the in vivo sample. GC content is represented as a filled orange curve. Different gene classes are separated by horizontal dotted lines. The nucleosome-depleted region upstream of standard nucleosomes coincides with GC-poor DNA. Pronounced peaks in GC content (orange arrows) exhibit a ∼200-bp periodicity, and coincide with nucleosome positions in vitro (blue arrows). This is consistent with GC-rich DNA being intrinsically favorable for nucleosome formation. Genes with no standard nucleosomes in vitro (top row) exhibit an indistinct nucleosome pattern in vivo (right panel). On the other hand, genes with a +1 nucleosome in vitro (blue arrow within left panel) exhibit increased nucleosome positioning in vivo, not only at the +1 position (red filled arrow), but also around this region (red open arrows). A model based on nucleosome sequence preferences successfully predicts in vitro nucleosome positions (black arrows), which in turn overlap with in vivo nucleosomes (red filled arrows). However, the model fails to predict in vivo nucleosomes in surrounding regions (red open arrows), suggesting that such nucleosomes are instead positioned by trans-acting factors. These trends are also observed in other gene classes, with varying numbers of nucleosomes in vitro. DNA sequences favorable for nucleosome formation may thus function as nucleation sites that aid trans-acting factors in positioning nucleosomes in flanking regions in vivo.

Figure 5.

Contrasting mechanisms may underlie conserved nucleosome patterns in vivo between Tetrahymena and yeast. The Tetrahymena genome is GC-poor and is generally unfavorable for nucleosome formation. The majority of Tetrahymena genes encode nucleosome-favoring sequences at subsets of standard positions downstream from TSSs, which might in turn facilitate nucleosome positioning in and around these regions in vivo. On the other hand, yeast genes generally show no such DNA-guided specificity near TSSs, instead relying mainly on trans-acting factors to generate the distinctive nucleosome organization in vivo. As a result, the average in vitro and in vivo nucleosome patterns appear similar in Tetrahymena but not yeast.