p53-induced Regulation of Transcription in the Chromatin Context

Zhurkin, Victor

Abstract

To elucidate DNA trajectory in the p53-DNA complex in solution, we used Iodine-125 radioprobing (in collaboration with I. Panyutin and R. Neumann, Clinical Center, NIH). This method is based on analysis of the DNA strand breaks produced by the decay of an electron-emitting radioisotope, Iodine-125, incorporated in the C5 position of cytosine. The weaker the DNA strand break the larger the distance from the radioisotope to the cleavage site. The major advantage of radioprobing is its applicability for very large protein-DNA complexes. In particular, this method allows direct comparison of the conformations of DNA bound to the p53 core domain and to the wild type protein, the latter still being beyond the scope of conventional methods such as crystallography and NMR. As to the CATG tetramers mentioned above, our data testify against formation of the Hoogsteen base pairs (in agreement with two x-ray structures out of three). Our results also indicate that in a tetrameric complex with wt p53, the central region of the consensus 20-bp DNA fragment (YYYRRR) is bent into the minor groove (that is, consistent with our model and with the p53 tetramer binding to nucleosome). The detailed visualization of the DNA trajectory requires more radioprobing data. In the near future, we anticipate obtaining such data for several DNA sequences, including those of the p53 REs activating cell cycle arrest and apoptotic genes (CCA and Apo-genes). Special attention will be paid to the dependence of DNA conformation on the length of a variable spacer S in the center of p53 RE. Comparing distribution of the p53 sites in the CCA- and Apo-genes, 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 because 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, 14-3-3sigma 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 (published in J. Biol. Chemistry). 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 CATG (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. We paid special attention to genome-wide distribution of putative p53 binding sites and their relationship to various transposable elements, in particular Alu repeats. First, we analyzed 160 functional p53 REs identified so far and found that 24 of them occur in repeats. More than half of these repeat-associated REs reside in Alu elements;they are located in the vicinity of Boxes A and B of the internal RNA polymerase III promoter. Interestingly, several Alu-residing p53 REs are associated with apoptotic genes (e.g., CASP-10). In addition, using a position weight matrix approach, we found approximately 400,000 potential p53 sites in Alu elements genome-wide. These sites are located in the same regions of Alu repeats as the functional p53 REs and thus can be divided into two groups depending on their vicinity to the Boxes A or B. The nucleosome-mapping experiments made on Alu elements earlier, suggest that the p53 sites from these two groups have different chromatin environments which is critical for the p53-DNA binding. Finally, we compared the p53 sites with the corresponding Alu consensus sequences and concluded that the two groups of sites probably evolved through different mechanisms one group was generated by CG-to-CA:TG mutations;the other group apparently pre-existed in the progenitors of several Alu subfamilies, such as AluSp and AluSx. Remarkably, this assessments holds both for the functional p53 REs and for putative BSs. Our observations may be important for understanding the evolution of the p53 regulatory network at the genome-wide level.