The high-throughput sequencing of 5′ capped sequence tags has clearly shown that eukaryotic promoters separate into at least two classes defined by focused and dispersed distributions of initiation events. Many recent studies have reported on the chromatin structure in eukaryotic genomes; our approach differed from most of these efforts by assessing chromatin features from the basis of transcription initiation as derived from 5′ tag data. In one exception, work concurrent to ours found differences on H3K9 acetylation based on different promoter classes 
. Here, we have established that promoters from different classes not only contain different core promoter sequence features, but also reflect distinct patterns of nucleosome organization, chromatin structure, and insulator preferences ().
Promoter Classes Are Indicative of Divergent Strategies for Transcription Initiation.
Our findings revealed that the periodic distribution of nucleosomes in the vicinity of TSSs was strongest for dispersed promoters (classes BP and WP), which have defined NFRs and highly periodic H2A.Z-containing nucleosomes. In contrast, focused promoters (class NP) exhibited significantly lower occupancy and/or less organized nucleosomes. Furthermore, recently defined insulator classes showed distinct associations: class I insulators (which include CTCF) were associated with H2A.Z organization and H3K4 methylation at WP promoters, whereas class II insulators were evenly distributed. Conversely, GAF and pol II showed higher levels at NP promoters. The enrichment of the Drosophila
GAF protein at NP promoters was intriguing, as it is a protein with many reported roles in transcription and chromatin remodeling 
, and may assist transcription initiation at NP promoters in the presence of unorganized nucleosomes. For instance, GAF forms a multimer in replacement of the NFR to establish proper nucleosome organization 
and is enriched at genes with polymerase stalling 
NP and WP promoters in fruit fly and human likely correspond to two classes of promoters that have been recently characterized in yeast 
. The first class has well-defined NFRs flanked by nucleosomes (Depleted Proximal Nucleosome, DPN), while the second class has variable nucleosome positioning without a clear NFR (Occupied Proximal Nucleosome, OPN). CAGE-like data is not available at a scale needed for the identification and assignment of promoter classes in yeast, but OPN promoters have a low association with H2A.Z, a high transcriptional plasticity, and are enriched for TATA boxes, while the opposite is true for DPN promoters. Our work supports and extends the yeast model, in which access to most eukaryotic focused/OPN promoters is highly regulated as the corresponding genes carry out specific functions in response to specific conditions, while expression from many dispersed/DPN promoters is constitutive because they perform housekeeping functions in the cell.
A separation of mammalian promoters has frequently been proposed based on the presence of CpG islands. Differential regulation of some promoters with CpG islands has been shown to result from unstable nucleosomes, contrary to the involvement of chromatin remodelers at non-CpG island promoters 
. Somewhat differently, we found that CpG islands are present across all initiation patterns, which indicates that CpG islands are not a homogeneous class and do not all encode constitutively unstable arrangements of nucleosomes. The work by Ramirez-Carrozzi et al. 
focused on a specific set of promoters, those adjacent to stimulus-response genes, in which nucleosomes are pre-organized to facilitate a regulated primary response. Such genes may form an intermediate class between constitutively expressed genes typically associated with CpG islands, and NP promoter genes, which contain genes like developmental TFs that are expressed in a precisely determined and highly regulated order. The conservation of our findings in Drosophila, as well as the previous studies in yeast, support that some CpG islands may provide an additional mechanism of sequence-encoded nucleosome propensities specifically found in mammals.
Multiple aspects may contribute to the relationship between the promoter classes and chromatin features. First, differences in chromatin architecture may be directly reflected in distinct initiation patterns, as illustrated by the nucleosome organization in constitutive versus regulated genes in yeast 
. Thus, in fly and human, dispersed promoters result from a well-defined NFR increasing the accessibility of the DNA to the polymerase, causing initiation to occur at multiple locations over a large region. In turn, the lower accessibility of focused promoters provides for a more regulated transcription initiation due to the lack of a common NFR. Instead, TSSs of focused promoters are well-defined by position-specific sequence elements including the canonical core promoter motifs 
, which serve to actively recruit the core complex to precise TSS locations. Our computational models clearly support this idea: chromatin features contribute to NP promoter definition, but much less so than for other classes, and with little improvement on sequence information. Overall, the higher pol II level at the TSSs of actively expressed genes with NP promoters also suggests that polymerase stalling is involved as an additional regulatory step enriched but not restricted to these genes 
Second, the relationship between the promoter classes and chromatin profiles may also be influenced by the duration of active transcription. It has been suggested that nucleosomes are properly positioned through repeated rounds of active transcription 
. As dispersed promoters, and focused promoters containing the TCT motif 
, are enriched in constitutively expressed genes 
, this would support the greater degree of nucleosome organization and the combinations of histone variants and chromatin marks (such as H2A.Z and H3K4me3) traditionally associated with active transcription. In turn, many focused promoters are associated with specific time points during embryogenesis 
, and the lack of constant transcription potentially leads to a reduced positioning of nucleosomes. Finally, promoters may have distinct chromatin patterns involving features we did not investigate. For instance, a higher rate of H3 turnover was observed at OPN promoters in yeast 
, and the presence of GAF has been associated with H3.3 replacement 
, suggesting the possibility that focused promoters may have a higher association with H3.3 replacement.
As more data becomes available through large-scale efforts such as the modENCODE and ENCODE projects, the presence of high-level divergent strategies of gene regulation established at the basal promoter will become better characterized throughout development and differentiation in model organisms as well as in human. Promoter classes may have associations to epigenetic inheritance, cellular memory, evolvability, and the development of disease 
. Understanding initiation patterns does not only help deepening our knowledge of the core promoter sequence, but also provide insight into the epigenetic architecture of regulatory regions. Together, they illustrate the interplay between chromatin and sequence information to encode divergent strategies for gene expression.