Comparing expressed and non-expressed genes, we observe several differences in nucleosome organization at the promoter regions. First, we observe increased nucleosome occupancy at expressed promoters when compared to non-expressed promoters. Interestingly, the increased amplitude is observed in most of the nucleosomes in the promoter region, with exception of the 21 nucleosome. We find that occupancy of the 21 nucleosome decreases at 6 hpf and 9 hpf at expressed promoters. Second, we detect changes in the spacing between the 21 and +1 nucleosomes of expressed and nonexpressed promoters. The larger spacing is most evident at 6 hpf and 9 hpf in the expressed promoters and coincides with a likely NDR. Due to this change in spacing, nucleosomes also appear out of phase between the expressed and non-expressed promoters. Finally, though hox transcription is dependent on RA signaling, we find that blocking RA signaling does not cause changes in nucleosome organization at the expressed promoters, suggesting that nucleosome arrangement is independent of Bortezomib RA-induced transcription. The fact that nucleosome organization is dynamic, but genomic sequence is invariant, during embryogenesis, also suggests that trans-factors play a role in dynamically positioning nucleosomes at the promoters of hox genes in the developing embryo. While these data suggest that transcription may have a direct effect on the nucleosome arrangement at hox promoters, we find that blocking RA signaling represses hox transcription with no changes in the nucleosome profile. We note that our DEAB protocol was designed to prevent initiation of hox transcription and that we may have observed a different effect if hox gene transcription had been allowed to initiate prior to being inactivated. Hence, our data suggest that the nucleosome profile at hox promoters is independent of RA-induced hox transcription. We see further support for this conclusion when Staurosporine 62996-74-1 embryos are treated with RA. Though exogenous RA induces hox transcription, RA-induced genes do not recapitulate the nucleosome positions observed at endogenously expressed promoters and display little change from nucleosome positions observed in untreated embryos, again suggesting that the nucleosome profile at hox promoters is independent of hox transcription. Our findings raise the question as to what role RA signaling plays in hox transcription if it does not affect nucleosome organization. Given the complexity of eukaryotic chromatin structure, it is possible that RA affects chromatin structure at a level distinct from the nucleosome. For instance, previous studies detected chromatin changes at the HoxB and HoxD clusters using fluorescent in situ hybridization. Hox loci were observed to decondense during mouse embryogenesis in correlation with hox gene transcription and this process was recapitulated by RAtreatment of ES cells. It is therefore possible that RA affects chromatin at the level of the 30 nm fiber without affecting the positioning of individual nucleosomes. It is also possible that RA affects hox expression by promoting histone modifications that are supportive of transcription. Indeed, RA receptors are known to recruit histone-modifying enzymes. Lastly, RA may simply recruit components of the transcription machinery, again via RA receptors, to hox promoters. The fact that RA induces hox transcription without affecting nucleosome organization could also be taken to indicate that many nucleosome arrangements are permissive for transcription. However, it is important to note that the exogenously applied RA is likely in significant excess relative to endogenous levels and this may permit over-riding of a nucleosome arrangement that would not otherwise support transcription.