Effect of Amino Acid Subsititution in Set1 on Histone H3 Methylation and Gene Silencing in Saaccharomyces Cerevisiae
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Chromosomal DNA in our cells is wrapped around a histone protein octomer like thread on a spool, forming a structure called a nucleosome. Series of nucleosomes form the nuclear chromosomes found in all eukaryotic organisms. Modifications to histone proteins can change how accessible chromosomal DNA is to protein complexes that act on DNA. DNA sequences that are inaccessible are called silent chromatin and regions that can’t be transcribed are subject to “gene silencing.” Proper gene silencing is necessary for normal cell development and regulation. Incorrect or missing histone modifications can cause the loss of gene silencing and uncontrolled gene expression similar to the situation in cells of patients with cancer or leukemia. My project focuses on a histone modifying complex COMPASS. COMPASS is composed of eight proteins, one of which is the histone H3 methyltransferase Set1. There are seven Set1 homologs in yeast and over 60 Set1-like proteins in humans, including MLL, which is known to be associated with human leukemia. Previous studies have shown that Set1 and most COMPASS proteins are essential for gene silencing at the ribosomal DNA locus (rDNA) in yeast. The SET domain is the active site of the Set1 histone methyltransferase, where methyl groups are covalently attached to the fourth lysine residue (K4) of histone H3. My goal is to investigate the effect of six individual amino acid substitutions in the SET domain of Set1; Y967A, I972A, Y993A, H1017L, Y967F, and G951A, on histone H3 methylation and gene silencing. These altered Set1 proteins are being expressed in the yeast Saccharomyces cerevisiae. Using Western blots and marker genes, the effect of these mutations are compared to wild type Set1. My data show that there are defects in histone H3 methylation in the amino acid substitution variants of Set1. In five of the mutants there is a complete loss of H3K4 methylation. In the future, we will determine if these altered Set1 proteins are assembled into the COMPASS complex. By characterizing the catalytic domain of Set1 using amino acid substitution variants, we will acquire a better understanding of the related proteins in humans.
Chateau, Morgan (2008). Effect of Amino Acid Subsititution in Set1 on Histone H3 Methylation and Gene Silencing in Saaccharomyces Cerevisiae. Available electronically from