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 assessment is further substantiated by a collaborative experimental study of the p53 tetramer binding to nucleosomal DNA (in preparation). According to our results, the p53 affinity to its cognate site strongly depends on the rotational positioning of this site in nucleosome. Namely, the p53-DNA binding is much more effective when the p53 RE is positioned in such a way that the tetramers CWWG (mentioned above) are bent into the major groove, and their minor groove is accordingly exposed. This is exactly the situation we envisioned in the case of the CCA-sites preformed for the p53 binding. Our model 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 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. Another study in progress is related to more general evolutionary aspects, such as the inter-relationship between the p53 REs in human genome and interspersed repeat sequences. First, we analyzed the genome-wide distribution of the spacer length, S, and found this to be highly non-random. (The spacer is inserted between two p53 half-sites, RRRCWWGYYY.) In particular, the p53 sites with S=0 and S=3 bp are nearly twice as frequent as the sites with S=2, 4 or 5 bp. Second, we showed that these differences are caused by transposons, in particular, by Alu repeats. In addition, we compared the spacer profiles for 2000 human genes known to be regulated by p53. It was found that the p53-activated genes are surrounded mostly by REs with S=0, whereas the promoter regions of p53-repressed genes are enriched with the p53 sites having S=3 bp. Importantly, this distinction becomes even more pronounced when only the genes with the strongest p53-induced effect are selected (manuscript in preparation). 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.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Intramural Research (Z01)
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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