Nuclear Envelope Directed Genome Organization in Myogenesis and Emery-Dreifuss Muscular Dystrophy

每个人的基因组在他们身体的所有细胞中都是相同的。然而，这些细胞能够实现令人难以置信的不同形式，从那些使心脏到肝脏或大脑。基因组调控有许多方面，使基因组中不同的基因子集能够表达，以帮助每个组织发育。其中最不为人所知的是基因在细胞核中的位置如何影响其调控。许多对组织发育很重要的基因随着它们的表达从细胞核的边缘向内部移动。其他对组织发育起拮抗作用的基因则移动到细胞核的边缘并被关闭。一般来说，位于核外围的基因往往会被关闭，有证据表明，核空间基因组组织的破坏是一些发育缺陷和疾病的基础。我们以前已经确定了位于细胞核边缘的蛋白质，这些蛋白质对建立空间基因组组织非常重要。这些蛋白质中的一些仅在肝脏中发现并影响肝脏中的基因组组织，而另一些仅在脂肪中发现并影响脂肪中的基因组组织，而另一些仅在肌肉中发现并影响肌肉中的基因组组织。我们在肌肉中发现了五种这样的蛋白质，不同的蛋白质影响了对肌肉发育重要的不同基因子集。在单独的工作中，我们研究了肌肉萎缩性疾病埃默里-德赖富斯肌营养不良症。已发现七种不同蛋白质的突变导致这种疾病，但临床诊断的Emery-Dreifuss患者中有一半以上没有这七种蛋白质的突变，因此必须有其他原因。我们分析了62名这样的Emery-Dreifuss患者，发现其中11人在五种基因组组织肌肉蛋白中的四种中存在突变。拟议项目的目标是测试发现的突变是否可以通过将突变工程化到一个细胞系统中来解释疾病的病理学，在这个细胞系统中，可以在培养皿和小鼠中跟踪肌肉发育。前者可以提供有关个体肌肉细胞何时何地发生特定缺陷的信息，而后者可以提供有关全身肌肉功能，代谢和病理学的信息。肌肉代谢标志物的水平将被测定，以及其他组织的标志物，以防突变的基因组组织蛋白未能关闭来自其他组织的基因，因为这可能有助于解释Emery-Dreifuss肌肉缺陷。肌肉大小，肌肉再生和肌肉干细胞功能也将进行分析。为了确定基因组组织缺陷是否可以解释细胞和组织病理学，将通过全基因组测序方法确定不同基因和其他基因组调控元件的表达，并使用显微镜观察正常与突变细胞和组织中基因的位置。上述研究将使用基因组组织蛋白和其他导致Emery-Dreifuss肌营养不良症的蛋白质中的突变体来完成，以确定潜在的缺陷是否匹配。最后，我们将通过对受影响基因周围的基因组进行特定修饰，并确定这些修饰是否阻止正常基因向核边缘或从核边缘移动，来研究基因组组织变化背后的机制。这项研究既可以深入了解基因组组织的基本机制，也可以确定Emery-Dreifuss肌营养不良症中改变的基因和代谢途径。这种综合理解可能会导致治疗方法专注于通过提供缺失的代谢物和酶来恢复适当的代谢平衡。

Each individual's genome is identical in all the cells of their bodies. Yet these cells are able to achieve incredibly different forms from those making the heart to the liver or brain. There are many aspects of genome regulation that enable different subsets of the genes in the genome to be expressed to help each tissue develop. One of the least understood of these is how the position of a gene in the nucleus contributes to its regulation. Many genes important for tissue development move from the edge of the nucleus to the interior concomitant with their being expressed. Other genes that are antagonistic to tissue development move to the edge of the nucleus and get shut down. In general the genes located at nuclear periphery tend to be shut down and there is evidence that disruption of nuclear spatial genome organisation underlies some developmental defects and diseases. We have previously identified proteins located at the edge of the nucleus that are important for establishing spatial genome organisation. Some of these proteins were found only in liver and affected genome organisation in liver, while others were found only in fat and affected genome organisation in fat and yet others were found only in muscle and affected genome organisation in muscle. We found five such proteins in muscle and different ones affected distinct subsets of genes important for muscle development. In separate work we were studying the muscle-wasting disease Emery-Dreifuss muscular dystrophy. Mutations in seven different proteins have been found to cause this disease, but a little over half of clinically diagnosed Emery-Dreifuss patients do not have mutations in these seven proteins and so must have other causes. We analysed 62 such Emery-Dreifuss patients and found that 11 of them have mutations in four of the five genome-organising muscle proteins. The goal of the proposed project is to test whether the mutations found can explain the pathology of the disease by engineering the mutations into a cell system where muscle development can be followed in a dish and also into mice. The former can give information about when and where specific defects occur in individual muscle cells while the latter can give information about systemic muscle function, metabolism and pathology. Levels of muscle metabolic markers will be assayed as well as markers of other tissues in case the mutant genome-organising proteins fail to shut down genes from other tissues as this could help explain Emery-Dreifuss muscle defects. Muscle size, muscle regeneration and muscle stem cell function will also be assayed. To ascertain whether genome organisation defects can explain the cell and tissue pathology, the expression of different genes and other genome regulatory elements will be determined by genome-wide sequencing approaches and the position of genes in normal versus mutant cells and tissues observed using microscopy. The above studies will be done both using mutants in the genome-organising proteins and in other proteins that cause Emery-Dreifuss muscular dystrophy to determine if the underlying defects match. Finally, we will investigate the mechanism behind the genome organisation changes by making specific modifications to the genome around the affected genes and determining whether these modifications block the normal gene movements to or from the nuclear edge. This study should both provide insights into the fundamental mechanisms underlying genome organisation and also identify the gene and metabolic pathways altered in Emery-Dreifuss muscular dystrophy. This combined understanding could lead to therapeutic approaches focused on restoring the proper metabolic balance by supplying missing metabolites and enzymes.