Maki Inada. Poster Presentation. "Linking the C-Terminal Domain Code of RNA Polymerase II to Modulating Chromatin States in fission yeast Schizosaccharomyces pombe”. Cold Spring Harbor Laboratory Eukaryotic mRNA Processing Meeting. Cold Spring Harbor, NY. August 2013.
Regulation of gene expression is essential for all living organisms. One critical step in modulating gene expression is altering the ability of the transcriptional enzyme, RNA Polymerase II (RNAPII), to access DNA by manipulating chromatin states. The carboxy-terminal domain (CTD) of RNAPII, is believed to play a critical role in chromatin remodeling through its recruitment of factors that modify histones. Conserved throughout evolution, the CTD contains a repeated Y1S2P3T4S5P6S7 heptapeptide sequence that undergoes dynamic posttranslational modifications. The capability of each serine in the sequence to undergo phosphorylation and dephosphorylation creates a readable ‘code’ for recruiting factors that can determine when processing events such as chromatin remodeling to occur. In order to characterize how specific phosphorylation marks in the CTD affect gene expression, mutants of fission yeast Schizosaccharomyces pombe were rendered defective for phosphorylation by substituting a nonphosphorylatable alanine in place of each serine in position 2 in the heptad sequence (S2A), each position 7 serine (S7A), or all serines in position 2 and position 7 in combination (S2A/S7A). In addition, a fourth mutant was created in which the position 7 serines were substituted for the phosphomimetic glutamic acid (S7E). We have performed microarray experiments with these mutants to study the genome-wide effects of eliminating and altering these phosphorylation events. In agreement with previous results, we have observed the expression of STE11, which is required for mating in S. pombe, to be low in the S2A mutant, whereas levels are restored in the double mutant S2A/S7A. However, while others have observed defects in snRNA levels with these mutants in human cells, we do not see a similar decrease with our S. pombe mutants. Interestingly, analyses of our microarray data reveals an upregulation of positionally related clusters of genes, specifically in some telomeric regions. Further quantitative PCR analysis of genes in these telomeric regions confirm a significant upregulation of gene expression in the telomeric regions spanning approximately 50kb. Our microarray analyses and subsequent qPCR validation suggest a role for the dynamic phosphorylation and dephosphorylation of serines within the CTD code in modulating the chromatin states in large telomeric regions of S. pombe.