Mechanisms for establishment and maintenance of allele specific expression of GATA2

控制基因表达是一个复杂和高度协调的过程，几乎每一个活的生物体都需要这个过程，并且已经表征了许多水平的基因调控。一个这样的水平被称为等位基因特异性表达（ASE），其中只有一个基因的两个拷贝用于创建蛋白质。 ASE反过来又受到DNA甲基化的影响，这一过程涉及通过一类称为DNA甲基转移酶的特定酶将甲基添加到DNA中的胞嘧啶残基上。ASE的生物学功能仍不清楚，可能涉及限制基因表达或影响ASE调节的蛋白质的相互作用。我们最近发现了一个基因，GATA 2，在细胞系CG-SH中表现出ASE。这项资助的总体目标是确定GATA 2等位基因特异性表达如何建立和维持的分子机制。为此，我们将从三个方面来看待这一现象。 首先，我们将检查其他细胞类型，看看我们是否可以识别出GATA 2显示出相同效果的任何其他细胞类型。 我们还将寻找与CG-SH中GATA 2周围的转录调控相关的其他蛋白质修饰（例如组蛋白甲基化），以了解其他蛋白质标记是否也与ASE相关。 其次，因为DNA甲基化被认为是ASE所必需的，我们将看看我们是否可以通过添加缺陷酶或使用影响DNA甲基化的药物来破坏CG-SH细胞中GATA 2的ASE。这些实验应该告诉我们哪些酶参与其中，以及GATA 2的ASE是否真的需要DNA甲基化。最后，我们将研究GATA 2表达水平或ASE的丧失是否会影响其正常的调节分化的能力，通过增加细胞中存在的GATA 2的数量和类型，同时诱导它们分化。研究这些特定目标的实验结果将为尚未完全理解的生物过程提供新的见解。 通过定义这一过程背后的分子机制，将有可能设计进一步的研究来调查这一现象在细胞分化过程中的意义。

Controlling gene expression is a complex and highly coordinated process that is required by virtually every living organism and numerous levels of gene regulation have been characterized. One such level is called allele specific expression (ASE), where only one of the two copies of a gene is used to create proteins. ASE is in turn influenced by DNA methylation, a process that involves the addition of a methyl group to cytosine residues in DNA by a specific class of enzymes called DNA methyltransferases. The biological function of ASE is still not clearly understood, and may involve limiting gene expression or influencing the interactions of the proteins regulated by ASE. We have recently discovered a gene, GATA2, that exhibits ASE in a cell line, CG-SH. The overall goal of this grant is to define the molecular mechanisms of how the allele specific expression of GATA2 is established and maintained. To do this we will look at three aspects of this phenomenon. Firstly, we will examine other cell types to see if we can identify any others where GATA2 shows this same effect. We will also look for other protein modifications (e.g. histone methylation) connected with transcriptional regulation around GATA2 in CG-SH to see if other protein marks are also associated with ASE. Secondly, because DNA methylation is believed to be required for ASE, we will see if we can disrupt the ASE of GATA2 in CG-SH cells by adding defect enzymes or by using drugs which affect DNA methylation. These experiments should tell us which enzymes are involved and if DNA methylation really is required for ASE of GATA2. Lastly, we will look at whether GATA2 expression levels, or the loss of ASE, influences its normal ability to regulate differentiation, by increasing the amount and type of GATA2 present in cells while inducing them to differentiate. The results of the experiments to study each of these specific aims will provide novel insight into a biological process which is still not completely understood. By defining the molecular mechanisms behind this process, it will be possible to design further studies to investigate the significance of this phenomenon in the process of cellular differentiation.