Since genes in this group will become expressed at later stages of embryogenesis, it is possible that this NDR forms in preparation for subsequent transcriptional activation. Nucleosome positioning has been shown to result from the combination of intrinsic characteristics of DNA sequence, such as base pair composition, and from factors that interact with DNA, such as transcription factors and ATP-dependent chromatin modifiers. However, the relative contribution of each mechanism remains unclear. A recent study addressed how cis-elements and trans-factors influence nucleosome positioning in yeast. By using YACs to transfer large DNA fragments SJN 2511 between divergent yeast strains, analysis of nucleosome organization in the native strain could be compared to nucleosome organization on the YAC in the new host strain. This analysis revealed that inter-nucleosome spacing and positioning of the +1 nucleosome was altered upon transfer to the new host strain. Since sequence remains constant between the YAC and native yeast strain, these findings suggest that trans-factors play a more important role in nucleosome positioning than cis-elements. Similarly, we find that nucleosome organization changes during embryogenesis, but since the underlying sequence is invariant during development, trans-factors also likely play a role in nucleosome positioning during embryogenesis. We note that the changes in nucleosome organization that we observe correlate with important transitions during embryonic development. In particular, 2 hpf and 4 hpf embryos display relatively disordered nucleosomes at promoter regions, while at 6 hpf and 9 hpf nucleosomes are readily identified. This change coincides with activation of the zygotic genome at the maternal zygotic transition, which occurs in a time window at approximately 3�C4 hpf. Our data do not reveal whether there is a causal relationship between this transition and the observed nucleosome rearrangement. However, since we observe better nucleosome positioning after the MZT, it is plausible that transfactors become expressed at the MZT and subsequently regulate nucleosome arrangements at hox promoters. Though the NDRs observed in our study are similar to other bulk studies, they are smaller than the NDR previously observed at human hox promoters, which was Gefitinib reported to be,500 bp. We suspect the difference in NDR lengths between the two studies is due to differences between zebrafish embryos and human cell lines. First, the embryo is made up of a heterogeneous population of cell types, while cell lines represent a homogeneous population. The heterogeneity of cell types in the embryo might lead to variable nucleosome occupancy. For instance, cells in the embryo that do not express a given hox gene might have a nucleosome positioned upstream of the TSS, thereby reducing the size of the NDR observed when signals from all cells in the embryo are averaged. Indeed, a previous study found nucleosomes to be differentially positioned at the serum albumin enhancer in a tissue specific manner in mouse. Such variable nucleosome occupancy presumably does not occur in cell lines since they represent a homogeneous population of cells that would all have similar nucleosome positions. Interestingly, if some cells in the embryo lacked the 21 nucleosome, then the NDR of these promoters would expand to 310 bp and 320 bp at 6 hpf and 9 hpf respectively, making it more similar to the NDR observed at hox promoters in human cell lines. Second, the difference in NDR length may be due to differences between fish and humans. For instance, divergence of regulatory sequences in the promoters as well as divergence in the trans-factors responsible for nucleosome positioning may lead to different sized NDRs. Support for this possibility comes from the analysis of NDRs in evolutionary divergent yeast species.