DNA Folding in Chromatin and Interaction with Transcription Factors

在2011-2012财年，我们继续努力阐明指导核小体旋转和平移定位的DNA序列模式。特别是，我们开发了一种新的DNA线程算法，正确地预测了体外精确定位的核小体的定位。我们还对许多双链DNA片段进行了全原子能量最小化处理，这些片段经历了类似于在结晶核小体中观察到的构象转变。这种结合的方法使我们向前迈出了重要的一步，朝着理解基因组DNA中加密的核小体密码迈进了一步。核小体中DNA的折叠伴随着相邻碱基对的侧向位移，这一点通常被忽略。然而，我们发现，剪切变形，称为滑动，在DNA折叠中发挥着比之前想象的更重要的作用。首先，在DNA的局部各向异性弯曲部位观察到的横向滑动变形定义了它在染色质中的超螺旋轨迹。其次，在核小体上变形DNA的计算成本是特定于序列的：在最优定位的序列中，最容易变形的碱基对步骤(CA：TG和TA)发生在大的正向滑动和负向滚动的位置(DNA在这些位置强烈弯曲或扭结到小槽中)。在这里，我们纳入了“真实”DNA的所有自由度，从而超越了传统模型的限制，忽略了碱基对的横向滑动位移。注意，我们的结果与体外序列选择(SELEX)实验非常一致。对不同GC含量序列的核小体定位的成功预测表明了我们基于DNA变形能计算的结构分析的潜在优势。在这方面，重要的是我们的核小体定位的基于知识的模型考虑到连接子组蛋白(LH)引起的序列特异性效应。LHS在核小体的进出点对富含AT的序列表现出较高的亲和力，这与富含AT的DNA在染色质中紧密紧凑的一般趋势是一致的。另一方面，富含GC的启动子往往缺乏核小体，因此很容易被转录机器访问。然而，当DNA甲基化时，情况就完全不同了。在这种情况下，尤其是核小体和一般染色质的稳定性增加，启动子变得更难获得，转录水平显著降低。甲基化诱导的肿瘤抑制基因沉默经常与人类癌症有关。我们将这种表观遗传效应与LHS的序列特异性属性联系起来，已知LHS不仅对富含AT的序列有更高的亲和力，而且对甲基化的DNA也有更高的亲和力。根据我们的模型，LHS通过主槽中的疏水作用与胸腺嘧啶和甲基化胞嘧啶结合。我们的初步结果证实了上述模型。通过突变核小体进出口点的DNA序列，我们证明了确实，在预测位置存在胸腺嘧啶簇确实增加了组蛋白H1-0与核小体的亲和力。我们的下一步是研究连接物组蛋白H1-0与核小体DNA的相互作用，使用羟基自由基和DNA的MNase/exoIII联合切割。我们将研究连接子中富含AT、GC和甲基化的DNA对H1-0结合的影响。在这一点上，重要的是连接子组蛋白的H1-0变体参与末端分化。我们预计，我们的努力可能有助于理解DNA甲基化引起的表观遗传效应的分子机制-特别是不同H1变体所扮演的角色。对体内核小体定位的分析表明，基因组中存在两种翻译定位信号：一种是物种独立的(通用的)，存在于所有已研究的核小体序列中，包括酵母、苍蝇、线虫和鸡。另一个信号是酵母特有的。我们正在根据核小体的现有晶体结构来解释这些新数据。

During the fiscal year 2011-2012, we extended our efforts to elucidate the DNA sequence patterns guiding rotational and translational positioning of nucleosomes. In particular, we developed a novel DNA threading algorithm correctly predicting positioning of nucleosomes precisely mapped in vitro. We also ran all-atom energy minimization of numerous double-stranded DNA fragments undergoing conformational transitions similar to those observed in crystallized nucleosomes. This combined approach allowed us to make an important step forward, toward understanding the nucleosome code encripted in genomic DNA. The folding of DNA in nucleosomes is accompanied by the lateral displacements of adjacent base pairs, which are usually ignored. We have found, however, that the shear deformation, called Slide, plays a much more important role in DNA folding than was previously imagined. First, the lateral Slide deformations observed at sites of local anisotropic bending of DNA define its superhelical trajectory in chromatin. Second, the computed cost of deforming DNA on the nucleosome is sequence-specific: in optimally positioned sequences the most easily deformed base-pair steps (CA:TG and TA) occur at the sites of large positive Slide and negative Roll (where the DNA strongly bends, or kinks, into the minor groove). Here, we incorporate all the degrees of freedom of 'real' DNA, thereby going beyond the limits of the conventional model ignoring the lateral Slide displacements of base pairs. Note that our results are in remarkable agreement with the in vitro sequence selection (SELEX) experiments. The successful prediction of nucleosome positioning for sequences of various GC-content demonstrates the potential advantage of our structural analysis, based on calculations of the DNA deformation energy. In this regard, it is important that our knowledge-based model of nucleosome positioning takes into account the sequence-specific effects caused by linker histones (LH). LHs demonstrate a higher affinity for the AT-rich sequences at the entry-exit points of nucleosomes, which is consistent with a general tendency of AT-rich DNA for a tight compactization in chromatin. On the other hand, the GC-rich promoters are often depleted of nucleosomes and thus are easily accessible for transcription machinery. The situation is quite different, however, when DNA is methylated. In this case, the stability of nucleosomes in particular, and of chromatin in general, is increased, the promoters become less accessible, and the level of transcription is significantly decreased. The methylation-induced silencing of tumor suppressor genes is frequently related to human cancer. We link this epigenetic effect with the sequence-specific properties of LHs, known to have a higher affinity not only for the AT-rich sequences, but also for the methylated DNA. According to our model, the LHs bind to thymines and methylated cytosines through hydrophobic interactions in the major groove. Our preliminary results confirm the model described above. Mutating the DNA sequence at the entry/exit points in nucleosome, we demonstrated that indeed, the presence of thymine cluster in the predicted position does increase the affinity of histone H1-0 to nucleosome. Our next step is to study interactions of the linker histone H1-0 with nucleosomal DNA using hydroxyl radical and combined MNase/exoIII cleavage of DNA. We will study the effects of AT-rich, GC-rich and methylated DNA in the linkers upon H1-0 binding. In this regard, it is important that the H1-0 variant of linker histone is involved in terminal differentiation. We anticipate that our efforts may help understanding the molecular mechanisms responsible for the epigenetic effects caused by DNA methylation - in particular, the roles played by different H1 variants. Analysis of the nucleosome positioning in vivo allowed us to demonstrate that there are two kinds of translational positioning signals in genome: one is species-independent (universal), inherent in all nucleosome sequences studied so far, including yeast, fly, nematode and chicken. The other signal is yeast-specific. We are interpreting these new data based on the available crystallographic structures of nucleosomes.