Mechanisms for establishment and maintenance of allele specific expression of GATA2

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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.**********