Exploring The Mechanism Underlying How Histone Methyltransferase SET2 Protein Level Is Regulated In Saccharomyces Cerevisiae

In Sacharomyces cerevisiae,Set2 is a RNA polymerase II(RNAPII)-associated histone methyltransferase which is responsible for multiple states of methylation(mono-,di-,and trimethylation)on histone H3 Lys36 residue(H3K36).Previous studies have demonstrated that Set2-mediated H3K36 methylation plays various important roles in modulating gene transcription,suppressing histone exchange and facilitating the maintenance of well-spaced chromatin structure.However,it's still elusive how Set2 itself is regulated both genetically and physically.BUR1 and BUR2 genes encode the catalytic and regulatory subunits of a cyclin-dependent protein kinase complex respectively.It has been reported that Bur1/Bur2 is closely related with Set2 genetically.Set2 protein level and H3K36 trimethylation are significantly reduced in BUR1 and BUR2 mutant stains.Thus,in this study,we will explore how Bur1/Bur2 regulates Set2 protein level,and whether the mechanism is different from the one that Ctk complex regulates Set2 in which Ctkl phosphorylates Ser2 of the C-terminal domain of RNAPII to facilitate an interaction with Set2.In addition,we will explore the potential E3 ligase responsible for Set2 degradation via the Synthetic Genetic Arrays(SGA)analysis.Our results indicated that a reduction H3K36me3 level is unable to be recovered in bur2A stains even when the levels of Set2 and phosphorylated Ser2 of RNAPII are rescued,indicating that the mechanism of how Bur1/Bur2 regulates Set2 is different from how Ctkl regulates Set2.Furthermore,we identified that Bur1/Bur2 kinase complex can directly phosphorylates Set2 using in vitro phosphorylation assay.Four phosphorylation sites were identified through mass spectrometry analysis.We showed that phosphorylation of Set2 by Bur1/Bur2 is required for the stability of Set2 and H3K36 methylation.Set2 protein level was recovered in the bur2?bre1? strain compared with the bur2? strain,and in vivo ubiquitination assay displayed multiple polyubiquitinated Set2 bands,which suggest that Brel is indeed the E3 ligase of Set2.These results establish a model about the upstream mechanism of Set2 in Saccharomyces cerevisiae.Our study provides more evidences for the mechanism of how H3K36 methylation is regulated,which may play an important role in related diseases.