The Role of the Set1 RNA Polymerase II Interacting Motif (SRIM) in Set1 Recruitment and Histone H3K4 Methylation

In eukaryotes, gene expression is regulated by epigenetic modifications. Among these modifications, histone H3 lysine 4 (H3K4) methylation has been associated with transcription activation. Set1 is the sole H3K4 histone methyltransferase in Saccharomyces cerevisiae and functions in a multi-subunit protein complex (Set1C) to catalyze H3K4 mono-, di-, and trimethylation. In addition, mis-regulation of Set1 human orthologs such as MLL (1 to 4) or SETD1A/B have been linked to a variety of cancers such as leukemia and early embryonic development defects. The current model indicates that the Paf1 complex (RNA polymerase II associated factor I) recruits Set1C to the phosphorylated CTD of RNA polymerase II (RNAPII) and chromatin, so that Set1C can deposit histone H3K4 methylation at actively transcribed genes. Here we show that the Set1 and RNAPII association is independent of Paf1 complex subunits. Our results also show that Set1 and RNAPII can interact independently of Set1-associated subunits. We have identified, for the first time, the region responsible for the Set1 and RNAPII interaction and have named this region the Set1 RNA polymerase II interacting motif (SRIM), which is in the uncharacterized region upstream of the previously identified Set1 RNA Recognition Motifs (RRM). Importantly, we have determined that Set1 interacts with the Ser5 phosphorylated form of CTD and that the SRIM is necessary for this interaction. Additionally, SRIM mutants show altered enrichment at mostly the 5’ regions of open reading frames of actively transcribed genes, including PYK1 and PMA1 and inducible genes, including PCK1. Moreover, one SRIM mutant shows a great decrease in H3K4 trimethylation levels globally and gene specifically. Interestingly, the deposition of global H3K4 mono- and dimethylation still occurs in the SRIM mutants. Together, these results suggest that the Set1 and RNAPII interaction is important for proper recruitment of Set1 and the deposition of H3K4 trimethylation to 5’ ends of actively transcribed genes, whereas Set1 could also be recruited to chromatin through other mechanisms independent of the Set1-RNAPII interaction to deposit H3K4 methylation. Furthermore, the loss of Set1 and SRIM mutants show sensitivity to 6-azauracil (6AU) indicating that the Set1 and RNAPII interaction is important for transcriptional elongation. Taken together, these data indicate that Set1 specifically interacts with the Ser5 phosphorylated form of RNAPII through the newly identified SRIM and that this interaction plays a role in the establishment of Set1-mediated H3K4 methylation to actively transcribing genes and inducible genes. In the meanwhile, we have constructed the “N-ICE” plasmids so that non-essential and essential genes can be N-terminally 3×FLAG tagged and expressed from an Inducible promoter (GAL1), Constitutive promoters (CYC1 or PYK1), or the Endogenous promoter. We have validated the N-ICE plasmid system by N-terminal tagging two non-essential genes (SET1 and SET2) and two essential genes (ERG11 and PKC1)