INFERRING CHROMOSOME ARCHITECTURE FROM GENOMIC SEQUENCE

真核生物染色体DNA通过周期性附着在蛋白质支架上而被分割成多个结构域。DNA上发生附着的位点，称为支架（或基质）附着区（S/MARs），强烈影响许多调控事件，包括转录增强和增强、DNA复制起始、逆转录病毒整合和细胞凋亡。由于S/MARs没有一致的相关序列，理解支架附着机制及其调控作用的进展受到了阻碍。本研究小组和我们的实验合作者最近的工作涉及真核支架结合中应力诱导的DNA双失稳（SIDD）。当研究者使用他的理论方法来计算含有已知S/MARs的超螺旋DNA序列的不稳定谱时，S/MARs位点与广泛不稳定的区域重合。我们的实验合作者已经证明，这些位点实际上经历了碱基分解，无论是在体外还是在体内。此外，SIDD的程度与结合强度高度相关。这项合作研究计划将扩展我们对支架附着和辅助调节事件中双相不稳定作用的研究。我们将详细研究SAR结合强度与SIDD特征之间的关系。我们将完整地描述SAR的SIDD属性，并根据我们对其所需属性的理解，使用这些信息来设计人工SAR元件。这些将由我们的合作者从原核DNA中构建，并评估它们的支架结合特性。我们将开发计算搜索策略，通过基因组DNA序列的SIDD特性来检测SAR元素。这将为直接从DNA序列推断染色体结构和核组织提供第一种方法。它将使许多关于基因组DNA的组织和调节的重要研究得以进行。它将用于开发新一代基因治疗的精确靶向和调控转染载体。

Eukaryotic chromosomal DNA is partitioned into domains by periodic attachment to a protein scaffold. The sites on the DNA where attachment occurs, called scaffold (or matrix) attachment regions (S/MARs), strongly influence many regulatory events including transcriptional augmentation and enhancement, initiation of DNA replication, retroviral integration, and apoptosis. Progress in understanding the mechanisms underlying scaffold attachment and its regulatory effects has been hampered by the fact that S/MARs have no consensus sequence associated with them. Recent work by this research group and our experimentalist collaborators implicates stress-induced DNA duplex destabilization (SIDD) in eukaryotic scaffold binding. When this investigator uses his theoretical method to calculate destabilization profiles of superhelical DNA sequences containing known S/MARs, the S/MAR sites coincide with regions of extensive destabilization. Our experimentalist collaborators have shown that these sites actually undergo base unpairing, both in vitro and in vivo. Moreover, the extent of SIDD is highly correlated with strength of binding. This collaborative research program will extend our investigation of the role of duplex destabilization in scaffold attachment and ancillary regulatory events. We will investigate in detail the associations between strength of SAR binding and SIDD characteristics. We will completely characterize the SIDD properties of SARs, and use this information to design artificial SAR elements based on our understanding of their required attributes. These will be constructed by our collaborators from prokaryotic DNA, and their scaffold binding properties will be assessed. We will develop computational search strategies to detect SAR elements in genomic DNA sequences by their SIDD properties. This will provide the first method for inferring chromosomal architecture and nuclear organization directly from the DNA sequence. It will enable a host of important studies to be performed regarding the organization and regulation of genomic DNA. It will be used to develop a new generation of precisely targeted and regulated transfection vectors for gene therapy.