The Set1 histone H3K4 methyltransferase complex: A mediator of gene expression, ergosterol homeostasis, and drug resistance

Paul Francis South, Purdue University

Abstract

The methylation of lysine 4 on histone H3 (H3K4) is a highly conserved post translational modification among eukaryotes. Largely associated with active gene transcription, histone H3K4 methylation has been an important focus in understanding the regulation of gene expression. With the identification of H3K4 specific methyltransferases that catalyze the addition of methyl groups on H3K4, and, more recently, demethylases that actively remove methyl groups, a highly dynamic system of regulation through the modulation of histone methylation is beginning to be understood. In fact, the methyltransferases and demethylases involved in H3K4 methylation have been linked to the regulation of cellular development; the disruption and overexpression of these enzymes can lead to human disease. Work by us and others have demonstrated that histone methyltransferases play important roles in the expression of various genes - including HOX genes - and other factors involved in development and differentiation. Although H3K4 methyltransferases play important roles in differentiation and gene expression, how these enzymes assemble, how they interact with chromatin, and how they are specifically involved in gene regulation xvii has not been completely described. My work on how protein-protein interactions affect yeast Set1 methyltransferase complex assembly and function attempts to address these questions and describes how a previously unidentified domain within the yeast Set1 complex member Bre2 is necessary for interaction with the complex member protein Sdc1. Interestingly, this domain-domain interaction is necessary for Bre2 and Sdc1 to assemble into the Set1 H3K4 methyltransferase complex, proper H3K4 methylation, and gene expression. More importantly, the human homolog of Bre2, ASH2L interacts with the Sdc1 homolog DPY-30 in a highly conserved manner. Furthermore, my work has attempted to understand the role of the SPRY domain and an uncharacterized N-terminus within Bre2 and ASH2L. The SPRY domain shows importance in interacting with chromatin and for assembly with the Set1 complex. The N-terminus looks to be important in assembly, and both domains are necessary for proper H3K4 methylation. In addition to understanding the role of specific domains and protein interactions within the Set1 complex, my work also focused on the biological importance of H3K4 methylation in the regulation of ergosterol biosynthesis. Previous studies have shown that Set1C member Bre2 is necessary for resistance to the drug Brefeldin A (BFA). My work shows that Set1 and methylation of H3K4 are also necessary for resistance to BFA. In determining the role of Set1 in BFA resistance, we discovered that Set1 is important for the expression of genes in the ergosterol biosynthetic pathway, including the rate limiting enzyme HMG-CoA reductase (Hmg1). Interestingly, deletion of SET1 also leads to a reduction in total cellular ergosterol. In addition, there is also an increase in the expression of DAN1 and PDR11, two genes involved in sterol uptake when Set1 is not expressed. As a consequence of the increased expression of uptake genes, sterols are actively taken up by the cells in aerobic conditions. Finally we show that loss of Set1's methyltransferase activity results in sterol uptake, and that when grown in the presence of ergosterol the set1Δ strain becomes resistant to BFA. Altogether, these studies provide insight into the role of Bre2 and Sdc1 in Set1 complex assembly and H3K4 methylation, as well as provide evidence that Set1 and H3K4 methylation are important for the regulation of ergosterol homeostasis and, as a result, resistance to the antifungal drug Brefeldin A.

Degree

Ph.D.

Advisors

Briggs, Purdue University.

Subject Area

Molecular biology|Genetics|Biochemistry

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