Book of Abstracts: Albany 2007
June 19-23 2007
The p53-DNA binding in the chromatin context: p53-induced activation of the apoptotic and cell cycle arrest genes
DNA is severely bent in the complex with p53 tetramer, as follows from the DNA cyclization (1), stereochemical analysis (2) and gel electrophoresis experiments (3). Recently, two dimeric p53-DNA complexes have been resolved crystallographically (4,5). In accord with our model (2,3), both structures demonstrate bending into the major groove in the central fragment CATG (see Figure). The overall directionality of DNA bending is such that the p53 tetramer can bind its cognate response element (RE) wrapped in nucleosome. This implies that in principle, structural organization of the p53 RE in chromatin can regulate its affinity to p53 ? for example, exposure of the DNA site in the bent conformation would facilitate the p53 binding.
We illustrate the functional importance of these findings by comparing the high affinity of p53 to the response elements associated with cell cycle arrest (CCA-sites), and the low affinity to RE associated with apoptosis (Apo-sites). To elucidate the molecular mechanisms of the p53-DNA binding selectivity, we examined the long-range genomic environment of the p53 response elements. 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. That is, the p53 binding to a ?distal? CCA-site and induction of the corresponding CCA-gene is 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 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 predicted rotational positioning of these nucleosomes implies that the p53 REs are exposed in the bent conformation similar to that shown in the Figure. Apparently, 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.
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. This dynamic environment would interfere with the p53 search for its cognate binding site and make 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 p53-DNA binding (increasing, in agreement with in vivo observations, the p53 affinity to the CCA-sites and decreasing its affinity to the apoptotic sites).
Laboratory of Cell Biology
Figure. (a) Directionality of the DNA bending in the nucleosome core particle, NCP (6) is consistent with that observed in the p53-DNA complex in solution (3) and in the p53-DNA co-crystals (4,5). The tetramers CATG and GATG are bent into the major groove (framed in the sequence scheme in the bottom). Note that the left half of the NCP 20-mer agrees with the p53 response element (RE) consensus sequence. The center of the NCP 20-mer (dashed line) is separated from the nucleosome dyad by 16 bp. (b) A model of the p53 tetramer interacting with the nucleosomal DNA. The p53 α-helices can penetrate into the DNA major groove without creating stereochemical clashes with the histones.
References and Footnotes