Positioned and G/C-capped poly(dA:dT) tracts associate with the centers of nucleosome-free regions in yeast promoters.

Eukaryotic DNA is packaged as chromatin: highly organized arrays of nucleosomes which profoundly affect the functions of the underlying sequence (Luger et al. 1997; Kornberg and Lorch 1999; Li et al. 2007). The recent emergence of genome-wide nucleosome maps in a variety of organisms have made it possible to study, at a global level, how nucleosome positioning emanates from sequence (Yuan et al. 2005; Albert et al. 2007; Lee et al. 2007; Kaplan et al. 2008; Mavrich et al. 2008a,b; Shivaswamy et al. 2008). Factors contributing to in vivo nucleosome positioning fall into two categories: intrinsic positioning effects arising from differential DNA–histone affinities imparted by sequence (Widom 2001) and extrinsic positioning effects not directly dependent on the quality of DNA–histone interactions. Intrinsic nucleosome positioning effects include 10-base pair (bp) periodic dinucleotides that contribute to rotational phasing (Ioshikhes et al. 2006; Segal et al. 2006), and poly(dA:dT) tracts that are thought to destabilize nucleosomes because of their intrinsic structure (Iyer and Struhl 1995; Suter et al. 2000). Extrinsic nucleosome positioning effects include passive exclusion (e.g., nucleosome occlusion by a bound transcriptional regulator) and active remodeling (e.g., nucleosome sliding/removal by ATP-dependent chromatin remodeling enzymes or transcriptional machines). Depending on genomic context, these intrinsic and extrinsic effects must be carefully balanced in order to achieve the nucleosome configurations necessary for biological function. Nucleosomes at yeast promoters have been extensively studied as paradigms of intrinsic and extrinsic positioning. At most yeast promoters, nucleosomes are highly ordered and are largely defined around a nucleosome-free region (NFR). The NFR appears to be a conserved mode of promoter nucleosome organization in most eukaryotes. NFRs are also closely related to the well-characterized DNase I hypersensitive sites in eukaryotes, where regulatory activity is manifested as local chromatin changes (Elgin 1981; Weintraub et al. 1981). In Saccharomyces cerevisiae, NFRs appear in up to 95% of promoters (Mavrich et al. 2008a) and typically span 140 bp (Lee et al. 2007; Shivaswamy et al. 2008). Furthermore, it has been shown that the conserved histone variant H2A.Z is heavily incorporated into the +1 and −1 nucleosomes flanking each NFR (Raisner et al. 2005). NFRs are organizing centers for the assembly of transcriptional machinery (Venters and Pugh 2009) and are thought to determine the locations of transcriptional start sites (TSSs) (Jiang and Pugh 2009). Mavrich et al. (2008a) have proposed a “barrier nucleosome” model of promoter chromatin organization, where strongly positioned +1 and −1 nucleosomes guide the positioning of flanking nucleosomes through statistical packing. This model accounts for the well-defined positions of NFRs, as well as the long-range order of nucleosomes, but requires robust mechanisms to correctly position the +1 and −1 “barrier nucleosomes” at each promoter. The question remains as to how the +1 and −1 nucleosomes are positioned, and whether this positioning occurs through intrinsic or extrinsic mechanisms or a combination of both. Mavrich and colleagues made the interesting observation that barrier nucleosome positions correlate with certain sequence features, such as nucleosome positioning sequence (NPS) and AA/TT dinucleotide gradient. However, the influences of NFR sequences and extrinsic nucleosome positioning factors on key promoter nucleosome positions have not been explored in detail. Extrinsic nucleosome positioning effects can be deconvoluted from intrinsic ones by comparative analysis of in vivo and in vitro nucleosome data. Such a study was recently performed (Kaplan et al. 2008) that found good overall correlation between in vivo and in vitro (assembled on purified genome DNA) nucleosome positions, suggesting that intrinsic DNA sequence preference is an important determinant of nucleosome positioning. We re-examined this data at finer granularity in promoters and found important differences between in vitro and in vivo nucleosome positions (Fig. 1). In many promoters, NFRs with sharply defined boundaries in vivo become very fuzzy in vitro, and the long-range ordering of nucleosomes flanking the NFRs is lost (Mavrich et al. 2008a). These observations suggest that active remodeling/transcriptional processes may be involved in shaping NFRs in the promoter regions. Figure 1. Comparison of in vitro and in vivo nucleosome maps at yeast promoters. (A) Nucleosome maps from tiling array data (Lee et al. 2007) for 2118 S. cerevisiae promoters were clustered using a self-organizing map (SOM) and visually partitioned into two groups ... While it is not fully understood how NFRs are specified by DNA sequence signals, poly(dA:dT) tracts—homopolymeric stretches of deoxyadenosine—are highly abundant in NFRs and have been strongly implicated as possible NFR positioning sequences (Behe 1995; Yuan et al. 2005; Lee et al. 2007; Segal and Widom 2009). Poly(dA:dT) tracts exhibit structural properties (Nelson et al. 1987; Woods et al. 2004), which could make them resistant to nucleosome incorporation and increase their accessibility to transcription factors (Iyer and Struhl 1995; Suter et al. 2000; Anderson and Widom 2001; Bao et al. 2006; Lee et al. 2007). In addition, computational approaches to nucleosome prediction have found that poly(dA:dT) tracts as short as length 3 have significant discriminative power in distinguishing nucleosomal versus non-nucleosomal sequence (Peckham et al. 2007; Yuan and Liu 2008). These studies suggest a role of poly(dA:dT) in the establishment of NFRs. In this study, we explore how poly(dA:dT) tracts contribute intrinsically and extrinsically to the formation of yeast NFRs by systematically analyzing the positions of these tracts relative to NFRs at a high resolution. We begin by comparing in vivo to in vitro nucleosome occupancy in promoters genome-wide (Kaplan et al. 2008). We found that promoters fall into two classes differing in the sharpness of NFRs in vivo. One class (class I) is enriched for TATA-less and constitutively expressed genes and shows stronger differences between in vivo and in vitro nucleosome occupancy patterns, compared to the latter class (class II). We then describe several novel characteristics of poly(dA:dT) tracts and show that these characteristics are correlated with NFR patterns unique to class I promoters. First, we show that poly(dA) and poly(dT) tracts have distinct localized spatial distributions in class I promoters, and that these distributions are symmetric across a central axis in the NFR. Second, we demonstrate that poly(dA:dT) tracts in NFRs exhibit an oriented, terminal specific “capping” by G:C. We demonstrate that the positions of G:C-capped poly(dA:dT) tracts are strongly correlated with NFR center positions. The localized and symmetric placement of poly(dA:dT) tracts in promoters, their orientation-specific base-capping characteristics, and their correlations with NFRs of class I promoters suggest that poly(dA:dT) tracts are important in directing NFRs in these promoters. Models of poly(dA:dT)-directed NFR formation and mechanistic implications are discussed.