Biophysical Models for Prediction and Design of Eukaryotic Chromatin Structure an

描述（由申请人提供）：我们的长期目标是预测分子相互作用如何转化为真核细胞中的基因表达。在真核生物基因组中，转录调控受到核小体的强烈影响，核小体的功能是压缩DNA，并通过简单的物理阻断和为许多共价表观遗传标签提供底物来调节对DNA的访问。我们最近开发了一种基于DNA力学的核小体模型，能够在体外以高精度再现核小体形成的自由能。我们建议将该模型应用于预测酿酒酵母和其他模式生物的全基因组核小体位置。我们将发展染色质结构的描述，包括组蛋白八聚体与其他dna结合因子竞争调控序列。我们的初步结果表明，这种竞争对于体内染色质结构的形成可能与内在核小体序列偏好一样重要。我们将通过探索模型系统中核小体位置和基因表达之间的联系来研究我们预测的准确性。我们建议构建启动子序列，将转录因子结合位点纳入计算设计的核小体占用谱中。这些结构将被用于基因表达水平的分析，为核小体发挥的调节作用提供直接的见解。此外，我们建议对体外重组的核小体进行高通量测序，包括基因组和化学合成的序列。该数据集将使我们能够从体内效应中分离出内在序列偏好，并使我们能够以系统的方式改进纯粹基于结构的DNA力学模型。最后，我们建议开展微阵列和大规模测序研究染色质结构对环境和遗传扰动的动态响应。所提出的研究将显著增强我们对调控DNA序列、染色质和基因表达之间联系的理解。

DESCRIPTION (provided by applicant): Our long-term objective is to predict how molecular interactions are translated into gene expression in eukaryotic cells. In eukaryotic genomes, transcriptional regulation is strongly affected by nucleosomes which function to compact DNA and to regulate access to it by simple physical occlusion and by providing the substrate for numerous covalent epigenetic tags. We have recently developed a DNA mechanics-based nucleosome model capable of reproducing in vitro free energies of nucleosome formation with high accuracy. We propose to apply this model to predicting nucleosome positions genome-wide, in S.cerevisiae and other model organisms. We will develop descriptions of chromatin structure that incorporate histone octamers competing with other DNA-binding factors for regulatory sequence. Our preliminary results indicate that this competition may be as important for shaping in vivo chromatin structure as intrinsic nucleosome sequence preferences. We will investigate the accuracy of our predictions by exploring the link between nucleosome positions and gene expression in model systems. We propose to construct promoter sequences that incorporate transcription factor binding sites into a computationally designed nucleosome occupancy profile. These constructs will be assayed for levels of gene expression, providing direct insight into the regulatory role played by nucleosomes. In addition, we propose to carry out high-throughput sequencing of nucleosomes reconstituted in vitro on both genomic and chemically synthesized sequences. This data set will allow us to disentangle intrinsic sequence preferences from in vivo effects, and will enable us to improve the purely structure-based DNA mechanics model in a systematic way. Finally, we propose to carry out microarray and large-scale sequencing studies of the dynamic response of chromatin structure to environmental and genetic perturbations. The proposed studies will significantly enhance our understanding of the connection between regulatory DNA sequence, chromatin, and gene expression. PUBLIC HEALTH RELEVANCE: The ability to predict how chromatin structure affects gene expression will open a novel pathway towards numerous applications in biology and medicine, including rational drug design and new, chromatin-based approaches to rewiring cellular networks. The ability to exercise precise transcriptional control over the amount and the type of proteins produced by the cell through making directed changes in chromatin structure will find many uses in bioengineering and synthetic biology, including production of synthetic hormones, enzymes, and therapeutic agents.