Genome Organization and Nuclear Architecture

在完成了对整个基因组进行测序的里程碑之后，现在理解人类生物学的基本问题是：基因组在活细胞中是如何组织的，以及决定哪些基因表达程序是活跃的机制是什么。我们对基因组和细胞核的细胞生物学研究旨在揭示体内基因组组织和核功能的基本概念，并为将这些原则应用于人类疾病诊断，治疗和生物工程提供机会。 为了了解基因组表达的核环境，我们正在使用体内成像来探测蛋白质和染色质的生物物理特性。为此，我们产生功能性的荧光标记分子，这些分子可以被引入细胞中，并通过延时显微镜在活细胞的细胞核中可视化。利用这些方法，我们已经成功地分析了活细胞中的基因表达过程，以研究哺乳动物细胞核的结构-功能关系。最近，我们已经开始研究，以测量生物物理性质的蛋白质在体内使用光漂白技术结合动力学建模和计算机模拟的方法。这些方法提供了强有力的工具，首次在分子水平上分析活细胞中蛋白质的作用，并在真实的时间。 理解基因组在细胞中如何发挥作用的第二个主要问题是确定基因组是如何组织的。为此，我们已经开始了研究，以映射的空间组织的染色体在细胞核内，我们正在测试是否定位的染色体影响基因的表达和调控。这些研究必须被认为是迈向严格间期细胞遗传学方法的第一步。 最后，我们正在使用我们的细胞生物学方法，通过研究前mRNA剪接因子与选择性剪接转录本的差异性关联来研究选择性剪接的细胞组织。我们还启动了一项研究，应用我们对选择性剪接位点选择的知识来纠正人类疾病基因的异常剪接，希望通过“RNA治疗”方法恢复正常剪接。

Having reached the milestone of sequencing entire genomes, fundamental questions in understanding human biology are now: How are genomes organized in living cells and what are the mechanisms that determine what gene expression programs are active. Our cell biological studies of genomes and the cell nucleus are aimed at uncovering fundamental concepts of genome organization and nuclear function in vivo and they are providing opportunities for applying these principles to human disease diagnosis, therapeutics and bioengineering. To understand the nuclear environment in which genomes are expressed, we are probing the biophysical properties of proteins and chromatin using in vivo imaging. To this end, we generate functional, fluorescently labeled molecules, which can be introduced into cells and visualized in the nucleus of living cells by time-lapse microscopy. Using these methods we have succeeded in analyzing gene expression processes in living cells to study the structure-function relationship of the mammalian cell nucleus. Recently we have initiated studies to measure biophysical properties of proteins in vivo by using photobleaching techniques in conjunction with kinetic modeling and computer simulation approaches. These methods provide powerful tools to analyze for the first time at the molecular level the action of proteins in living cells and in real time. The second major issue in understanding how genomes function in cells is to determining how genomes are organized. To this end we have initiated studies to map the spatial organization of chromosomes within the nucleus and we are testing whether positioning of chromosomes affects gene expression and regulation. These studies must be considered first steps towards rigorous interphase cytogenetic methods. Finally, we are using our cell biological approaches to investigate the cellular organization of alternative splicing by investigating the differential association of pre-mRNA splicing factors with alternatively spliced transcripts. We have also initiated a study to apply our knowledge of alternative splice site selection to correct aberrant splicing in human disease genes with the hope to restore normal splicing in a 'RNA-therapy' approach.