DNA Folding in Chromatin and Interaction with Transcription Factors

染色质中的 DNA 折叠及其与转录因子的相互作用

• 批准号: 8552858
• 负责人: Victor Zhurkin
• 金额: $ 71.42万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8552858
• 关键词: AT Rich Sequence; Accounting; Affinity; Agreement; Algorithms; Anisotropy; Base Pairing; Binding; Cell physiology; Chickens; Chromatin; Chromatin Loop; Code; Collaborations; Combined Modality Therapy; Cytosine; DNA; DNA Folding; DNA Methylation; DNA Sequence; DNA Sequence Rearrangement; Databases; Dependence; Digestion; Epigenetic Process; Eukaryota; Exodeoxyribonuclease III; Freedom; Genetic Transcription; Genome; Genomics; Goals; Guanine + Cytosine Composition; Histone H1; Histone H1(s); Histones; Human; Hydrophobic Interactions; Hydroxyl Radical; In Situ; In Vitro; Lateral; Link; Linker DNA; Major Groove; Malignant Neoplasms; Maps; Methylation; Micrococcal Nuclease; Minor Groove; Modeling; Modification; Molecular; Mutate; National Institute of Child Health and Human Development; Nematoda; Nucleosomes; Pattern; Play; Positioning Attribute; Property; Proteins; Relative (related person); Resolution; Roentgen Rays; Role; Running; Sequence Analysis; Signal Transduction; Site; Slide; Solutions; Structure; Thymine; Transcriptional Activation; Tumor Suppressor Genes; Variant; Yeasts; base; conformational conversion; cost; ds-DNA; fly; genome-wide; in vivo; knowledge base; novel; preference; promoter; research study; retinal rods; stem; transcription factor

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.

在2011-2012财政年度，我们扩大了我们的努力，以阐明DNA序列模式指导核小体的旋转和平移定位。特别是，我们开发了一种新的DNA线程算法正确预测定位的核小体精确映射在体外。我们还运行了所有原子能量最小化的许多双链DNA片段进行构象转变类似于那些在结晶核小体中观察到的。这种结合的方法使我们朝着理解基因组DNA中的核小体密码迈出了重要的一步。核小体中DNA的折叠伴随着相邻碱基对的侧向位移，而这些通常被忽略。然而，我们已经发现，剪切变形，称为滑动，在DNA折叠中起着比以前想象的更重要的作用。首先，在DNA局部各向异性弯曲的位点处观察到的横向滑动变形定义了其在染色质中的超螺旋轨迹。其次，计算出的核小体上DNA变形的代价是序列特异性的：在最佳定位的序列中，最容易变形的碱基对步骤（CA：TG和TA）发生在大的正滑动和负滚动的位点（DNA强烈弯曲或扭结到小沟中）。在这里，我们将所有的自由度的“真实的”DNA，从而超越了传统的模型的限制，忽略了横向滑动位移的碱基对。请注意，我们的结果与体外序列选择（SELEX）实验非常一致。成功预测的核小体定位的序列的各种GC含量证明了我们的结构分析的潜在优势，基于计算的DNA变形能。在这方面，重要的是，我们的核小体定位的知识为基础的模型考虑到连接组蛋白（LH）引起的序列特异性的影响。LH在核小体的入口-出口点处对富含AT的序列表现出更高的亲和力，这与富含AT的DNA在染色质中紧密致密化的一般趋势一致。另一方面，富含GC的启动子通常耗尽核小体，因此易于被转录机器接近。然而，当DNA被甲基化时，情况就完全不同了。在这种情况下，特别是核小体的稳定性，以及一般染色质的稳定性增加，启动子变得更难接近，并且转录水平显著降低。甲基化诱导的肿瘤抑制基因沉默通常与人类癌症有关。我们将这种表观遗传效应与LH的序列特异性特性联系起来，已知LH不仅对富含AT的序列具有更高的亲和力，而且对甲基化DNA也具有更高的亲和力。根据我们的模型，LHs通过大沟中的疏水相互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。在核小体的入口/出口点突变DNA序列，我们证明，确实，在预测位置的胸腺嘧啶簇的存在确实增加了组蛋白H1-0对核小体的亲和力。我们的下一步是使用羟基自由基和组合的MNase/exoIII切割DNA来研究连接体组蛋白H1-0与核小体DNA的相互作用。我们将研究接头中富含AT、富含GC和甲基化的DNA对H1-0结合的影响。在这方面，重要的是接头组蛋白的H1-0变体参与终末分化。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变体所起的作用。通过对核小体在体内定位的分析，我们发现基因组中存在两种翻译定位信号：一种是种间无关的（通用的），存在于所有已研究的核小体序列中，包括酵母、蝇、线虫和鸡。另一个信号是酵母特异性的。我们正在根据核小体的现有晶体学结构解释这些新数据。