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并通过简单的物理封闭和通过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，能够高精度地再现体外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。我们将开发染色质结构的描述，其中包含与其他DNA结合因子竞争调节序列的组蛋白八聚体。我们的初步结果表明，这种竞争可能是重要的塑造在体内染色质结构的内在核小体序列偏好。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有率。这些构建体将被测定基因表达水平，提供对核小体所起的调节作用的直接洞察。此外，我们建议进行高通量测序的核小体重建在体外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解开内在序列偏好，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，我们建议进行微阵列和大规模测序研究的动态响应的染色质结构的环境和遗传扰动。拟议的研究将显着提高我们的理解调控DNA序列，染色质和基因表达之间的联系。 公共卫生相关性：预测染色质结构如何影响基因表达的能力将为生物学和医学中的许多应用开辟一条新的途径，包括合理的药物设计和基于染色质的新方法来重新连接细胞网络。通过直接改变染色质结构对细胞产生的蛋白质的量和类型进行精确转录控制的能力将在生物工程和合成生物学中找到许多用途，包括合成激素，酶和治疗剂的生产。

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.