A functional evolutionary approach to identify determinants of nucleosome positioning: a unifying model for establishing the genome-wide pattern

Abstract

Although the genomic pattern of nucleosome positioning is broadly conserved, quantitative aspects vary over evolutionary timescales. We identify the cis and trans determinants of nucleosome positioning using a functional evolutionary approach involving S. cerevisiae strains containing large genomic regions from other yeast species. In a foreign species, nucleosome depletion at promoters is maintained over poly(dA:dT) tracts, whereas internucleosome spacing and all other aspects of nucleosome positioning tested are not. Interestingly, the locations of the +1 nucleosome and RNA start sites shift in concert. Strikingly, in a foreign species, nucleosome-depleted regions occur fortuitously in coding regions, and they often act as promoters that are associated with a positioned nucleosome array linked to the length of the transcription unit. We suggest a three-step model in which nucleosome remodelers, general transcription factors, and the transcriptional elongation machinery are primarily involved in generating the nucleosome positioning pattern in vivo.

Figure 1. Functional evolutionary dissection of chromatin establishment mechanisms

(A) Schematic of experimental design. Yeast Artificial Chromosomes are constructed carrying sequence from species such as K. lactis, and introduced into S. cerevisiae. Comparison of nucleosome mapping data between the same sequence in two different environments (its endogenous genome, and in S. cerevisiae) can be used to disentangle DNA-driven from trans-mediated aspects of chromatin organization. (B) Chromosomal complement of parental S. cerevisiae (AB1380) and 3 different YAC-bearing strains. Pulsed field gel electrophoresis of YAC-bearing strains, as indicated. (C–D) Examples of nucleosome mapping data from two genes. Blue line indicates nucleosome mapping data from wild-type K. lactis (Tsankov et al., 2010), red line shows data from the same sequence carried on a YAC in S. cerevisiae. (E–F) Data for all K. lactis genes on all 3 YACs. (E) shows data for all genes from wild-type K. lactis, with genes sorted by NDR width, while (F) shows data from these genes on YACs, sorted identically. Black indicates no sequencing reads, yellow intensity indicates number of sequencing reads. C and D indicate the example genes shown above.

Figure 2. Promoter nucleosome depletion is maintained over poly(dA:dT) elements

(A) D. hansenii genes sorted by the extent of change in nucleosome occupancy over the NDR. Left panel shows differences in nucleosome occupancy between D. hansenii and YACs for 114 genes – blue indicates increased nucleosome occupancy in the YAC relative to endogenous context. Middle and right panels show nucleosome mapping data for endogenous D. hansenii sequences and for YACs, as indicated. (B) Strength of poly(dA:dT) element (Field et al., 2008; Tsankov et al., 2010) for genes, ordered as in (A). 40 gene running window average is shown. (C) An example of a gene with little change in nucleosome depletion between endogenous and YAC contexts. Sequence from this stable NDR contains multiple poly(dA:dT) elements, as indicated in red. (D) An example of a gene exhibiting dramatically increased nucleosome occupancy at the native NDR when carried on YAC. Here, this NDR includes few polyA elements, and carries a binding site for Cbf1, which has nucleosome-evicting activity in D. hansenii but not in S. cerevisiae (Tsankov et al., 2011; Tsankov et al., 2010).

Figure 3. Nucleosome spacing is set in trans

(A) Averaged data for all K. lactis genes on YACs 1–3. Genes are aligned by the +1 nucleosome position as defined in Tsankov et al., and data from either wild-type K. lactis or from the YAC strains are averaged for 184 genes, as indicated. (B) K. lactis sequences adopt S. cerevisiae spacing when carried in S. cerevisiae. Nucleosome positions were called, and the distribution of all internucleosomal distances (center to center) is shown for 184 K. lactis genes from wild-type or in the YACs. Similar distributions for S. cerevisiae nucleosome positioning from wild-type and YAC-containing strains indicates that YACs do not perturb host chromatin state (See also Figure S2).

Figure 4. +1 nucleosome shifts associated with transcriptional changes

(A–B) Nucleosome data and RNA-Seq data are shown for K. lactis and D. hansenii genes in wild-type and YACs, as indicated. RNA-Seq data for YAC-derived transcripts are normalized independently from S. cerevisiae transcripts here – see Figures S4B–C for data normalized genome-wide. (C–E) Examples of +1 nucleosome shifts associated with changes in transcription. (C) shows a moderate upstream shift in a +1 nucleosome with a similar change in transcript length, while (DE) show large scale NDR gain/loss with associated changes in transcription. Schematic interpretation of the nucleosome positioning for the endogenous gene is shown in blue above the rectangle, nucleosome positioning in the YAC is shown in red below the rectangle. Arrows indicate inferred TSSs (note that RNA-sequencing data are not strand-specific, but TFIIB mapping data support our inferred TSSs) – the furthest 5′ RNA in (E), for example, derives from the upstream gene as opposed to a divergent promoter.

Figure 5. Characterization of fortuitousNDRs in YACs

(A) Example of a fortuitous NDR that occurs only in the YAC but not in the native genome, and is associated with transcription. This fortuitous NDR occurs in the middle of a D. hansenii coding region, and is associated with two shorter, divergent transcripts in the YAC context (data cover 2.2 kb of sequence). Note that nucleosome organization correlates with transcript length – rightmost transcript shows greater nucleosome positioning at the 5′ end than at the 3′ end of the transcript. (B) Fortuitous NDRs are generally associated with well-positioned +/−1 nucleosomes. Averaged data for 120 NDRs observed in D. hansenii YACs but not in the endogenous context, as indicated. (C–D) Fortuitous NDRs represent functional promoters. (C) shows TFIIB ChIP-Seq data from YAC-bearing strain for the genomic locus shown in (A), while (D) shows averaged data for all fortuitous NDRs. Note that TFIIB localization in the endogenous context could not be obtained as our anti-TFIIB antibody does not recognize TFIIB from D. hansenii.

Figure 6. Three-step model for establishment of nucleosome positioning in vivo

A unifying three-step model for how nucleosome positioning pattern is generated in eukaryotic organisms. The first step is the generation of an NDR, either by poly(dA:dT) elements and/or by transcription factors and their recruited nucleosome remodeling complexes. In the second step, nucleosome-remodeling complexes recognize the NDRs and generate highly positioned nucleosomes flanking the NDR; and the RNA polymerase II preinitiation complex fine-tunes the position of the +1 nucleosome. In the final step, positioning of the more downstream nucleosomes depends on transcriptional elongation, and the recruitment of nucleosome-remodeling activities and histone chaperones by the elongating RNA polymerase II machinery.