Unexpectedly, we found that the CCA-sites are located 2-3 kb away from the transcription start sites (TSS) of the target genes, whereas most of the Apo-sites are clustered within 1 kb from TSS. Note that such a distribution of the p53 sites is counter-intuitive contrary to our naive expectations, the p53 binding to a distal CCA-site and induction of the corresponding CCA-gene appears to be more efficient than the p53 binding to a close Apo-site and activation of the Apo-gene. We further showed that the flanking sequences of the CCA-sites, with moderate or low GC content (35-55 % GC), reveal strong periodicity of the AT-rich and the GC-rich clusters, similar to that observed in the nucleosomal DNA sequences, suggesting that stable positioned nucleosomes are likely to form here. (The limited experimental data available for several CCA-sites p21, 14-3-3σ and GADD45 are consistent with this assessment.) The predicted rotational positioning of these nucleosomes implies that the p53 REs are exposed in the bent conformation favorable for the p53 recognition. To put it differently, the bendable DNA elements in the vicinity of the CCA-sites are organized in such a way that the nucleosomal DNA is preformed for the p53 tetramer binding. For example, the p21 5-response element, the most effective p53 RE in vivo, is separated from TSS by 2.5 kb, and is bent in the same favorable conformation as observed in the crystallized nucleosomes. We suggest that exposure of the p21 and other CCA-sites accelerates the process of p53 binding in vivo. p53, in turn, recruits co-activators such as p300/CBP and/or chromatin remodeling factors to the promoters, thereby facilitating opening of chromatin and increasing the level of transcription. (The detailed molecular mechanisms of this long-distance transfer are not known. The enhancer-type looping of the higher-order chromatin fibril is a likely possibility. In such a case, the long distance between the strong CCA-sites and TSS would be a natural consequence of the chromatin rigidity looping of 2-3 kb fibril is much more favorable energetically than looping of 0.5-1 kb.) By contrast, the Apo-sites are located in extremely GC-rich regions (up to 75-80 % GC). Such sequences are typically characterized by multiple positioning and relatively easy reorganization of nucleosomes, as well as low H1 level. We hypothesize that this dynamic environment interferes with the p53 search for its cognate binding site and makes it less effective. Thus, the difference in nucleosomal organization of the two sets of p53 response elements appears to be a key factor affecting the strength of p53-DNA binding and kinetics of induction of the p53 target genes. Our interpretation differs from the earlier concept connecting the selective activation of the CCA- and Apo-genes to the binding affinities of their REs to p53. Instead, we emphasize a direct correlation between the selection of p53-induced tumor suppression pathway (apoptosis versus cell cycle arrest) and structural organization of the corresponding p53-binding sites in chromatin. We add new dimensions to the existing paradigm the relative positioning and chromatin environment of the p53 REs. Our scheme not only explains the above cases but also provides a new insight into the cellular mechanisms of activation of hundreds (if not thousands) of genes by p53. For example, its intriguing to see whether our simple model has a more general significance, beyond the limit of the CCA- and Apo-genes. To this aim, we compared several hundred human genes revealing various kinetics of the p53-induced activation in the same type of experiments. Specifically, we asked whether the p53-induced genes with early response differ from those with late response in terms of positioning of their REs relative to TSS. For most of these genes, the functional p53 REs are unknown, therefore, we analyzed positioning of the putative p53 binding sites predicted by the bioinformatic tool developed by us earlier. The distribution of hypothetical p53 sites is similar to that described above: the genes with early response have p53 sites located 2-3 kb away from the promoter region, while for the genes with late response, p53 sites are mostly within 1 kb from TSS. Thus, we suggest that the genomic environment of the p53 binding sites (in particular, the chromatin organization of the flanking sequences) is an important element in a general mechanism of orderly trans-activation of the p53 target genes. In the future, we intend to widen the scope of our research, and compare the early- and late-response genes activated by NF-κB and glucocorticoid receptor (GR) regulation of these genes has been intensively studied; in particular, there is a large set of GR-activated genes, with various kinetic profiles of activation. (Note that both these transcription factors bind to DNA wrapped in nucleosome; in this regard, NF-κB and GR are similar to p53.) The results will give us a better understanding of the relationship(s) between the mechanism(s) of gene regulation and the genomic environment of the TF binding sites operative in this regulation

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National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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Shen, Wen Hong; Wang, Jianli; Wu, Jingjing et al. (2006) Mitogen-activated protein kinase phosphatase 2: a novel transcription target of p53 in apoptosis. Cancer Res 66:6033-9