p53-induced Regulation of Transcription in the Chromatin Context

出人意料的是，我们发现CCA位点距离靶基因的转录起始点(TSS)2-3kb，而大多数Apo位点聚集在TSS的1kb以内。注意，这种P53位点的分布与我们天真的预期相反，P53与远端CCA位点结合并诱导相应的CCA基因似乎比P53与接近的Apo位点结合并激活Apo1基因更有效。我们进一步表明，CCA位点的侧翼序列，具有中等或低GC含量(35-55%GC)，显示出富AT和富GC簇的强烈周期性，与核小体DNA序列中观察到的相似，表明这里可能形成稳定定位的核小体。(几个CCA站点p21、14-3-3和#963；以及GADD45的有限实验数据符合这一评估。)预测的这些核小体的旋转定位表明，P53 RES暴露在有利于识别P53的弯曲构象中。换句话说，CCA位点附近的可弯曲DNA元件是这样组织的，即核小体DNA是为P53四聚体结合而预先形成的。例如，p215反应元件，体内最有效的p53 RE，与TSS分离了2.5kb，并以与在结晶核小体中观察到的相同的有利构象弯曲。我们认为，p21和其他CCA位点的暴露加速了体内P53结合的过程。P53反过来招募共激活因子，如p300/CBP和/或染色质重塑因子到启动子，从而促进染色质的打开，提高转录水平。(这种长距离转移的详细分子机制尚不清楚。高阶染色质原纤维的增强子类型环是一种可能的可能性。在这种情况下，强CCA位点和TSS之间的长距离将是染色质刚性环的自然结果，2-3kb的原纤维在能量上比0.5-1kb的环更有利。)相比之下，载脂蛋白位点位于GC含量极高的区域(高达75%-80%的GC)。这种序列的典型特征是多个定位和相对容易的核小体重组，以及低H1水平。我们假设，这种动态环境干扰了P53对其同源结合位点的搜索，使其效率降低。因此，这两组P53反应元件在核小体组织上的差异似乎是影响P53-DNA结合强度和P53靶基因诱导动力学的关键因素。我们的解释不同于早期的概念，将CCA基因和Apo基因的选择性激活与其RES与P53的结合亲和力联系起来。相反，我们强调选择P53诱导的肿瘤抑制途径(细胞凋亡与细胞周期停滞)与染色质中相应的P53结合部位的结构组织之间存在直接关联。我们给现有的范例增加了新的维度，即P53 RES的相对定位和染色质环境。我们的方案不仅解释了上述情况，还为P53激活数百(如果不是数千)基因的细胞机制提供了新的见解。例如，看看我们的简单模型是否具有更普遍的意义，是否超越了CCA和Apo基因的限制，这是很有趣的。为此，我们在同一类型的实验中比较了数百个人类基因，揭示了P53诱导激活的不同动力学。具体地说，我们询问了P53诱导的早期反应基因和晚期反应基因在其RES相对于TSS的位置上是否有所不同。对于这些基因中的大多数，功能上的P53 RES是未知的，因此，我们分析了我们先前开发的生物信息学工具预测的P53结合位点的定位。假想的P53位点的分布与上述相似：早期反应基因的P53位点距离启动子区域2-3kb，而晚期反应基因的P53位点大多位于TSS的1kb以内。因此，我们认为P53结合位点的基因组环境(特别是侧翼序列的染色质组织)是P53靶基因有序反式激活的一般机制中的重要因素。在未来，我们打算扩大我们的研究范围，比较由核因子-B和糖皮质激素受体(GR)激活的早反应和晚反应基因；特别是糖皮质激素受体(GR)对这些基因的调控已经得到了深入的研究；特别是GR激活的基因集，具有不同的激活动力学曲线。(请注意，这两种转录因子都与包裹在核小体中的DNA结合；在这一点上，NF-B和GR类似于p53。)这一结果将使我们更好地理解(S)基因调控机制(S)与作用于这种调控的Tf结合位点的基因组环境之间的关系

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