Mechanistic Study Of The Expression Of Histone Methyltransferase Set1

In S.cerevisiae,methylation on histone H3K4 is mediated by the Set1/COMPASS complex.Set1/COMPASS is a complex composed of eight proteins that can catalyze H3K4methylation.Set1 complex is highly conserved from yeast to humans.In addition to the catalytic C-terminal SET domain,Set1 has a conservative RNA recognition motif.As the only H3K4 methyltransferase in yeast,Set1 plays an important role in cell growth,transcriptional activation,telomere silencing,mitosis,DNA damage repair and chromosome separation.Previous research in our lab has shown that Set1 can significantly facilitate histone gene transcription to extend cell lifespan and maintain telomere silencing.As histone gene transcription is strictly regulated by cell cyle,it is possible that the expression of Set1 could also be regulated by cell cycle.In this thesis,we studied how cell cycle regulates Set1expression.By synchronizing the yeast cells in different cell cycles,we found that the protein level of Set1,but not the transcription level,was reduced significantly within the cell cycle.Our study further demonstrated that Set1 is degraded by the proteasome pathway and uncovered the underlying mechanism.The specific research contents are as follows:1.Yeast cell cycle was synchronized at different phases with drugs（α-factor,HU,nocodazole）,and Set1 protein level was examined by Western blots.The Set1 protein level was significantly reduced during G1 phase.According to the literature,APC/C^Cdh1（late promotion complex）specifically regulates the level of G1 cyclin in the cell cycle.Therefore,we screened the subunits of APC/C and activator deletion mutants in yeast deletion mutant library.Our results showed that the Set1 protein level recovered significantly in G1 phase after deletion of activator CDH1 and APC/C subunit APC9,APC10 and APC13,when compared with WT.Therefore,Cdh1 and APC/C subunits Apc9,Apc10 and Apc1 are key activators and subunits of the APC complex that recognize and degrade Set1 in G1 phase.By analyzing the sequence of Set1,we identified the existence of D-box,which can be recognized by APC/C^Cdh1complex.We therefore mutated the D-box within Set1（Set1-KNSA）and found that the APC/C^Cdh1complex could not degrade the Set1-KNSA mutant in G1 phase.2.By analysis of the D-box of Set1,we found that S228 within D-box could be a potential phosphorylation site.We then confirmed S228 phosphorylation using the specific antibody.Using protein mass spectrometry,we found that Set1 interacts with protein kinases Cla4.Knockout of CLA4 significantly reduced Set1 protein levels.Further study showed that Cla4 phosphorylates Set1-S228 to prevent Set1 degradation by APC/C^Cdh1complex.3.Cla4-catalyzed Set1 phosphorylation promotes histone gene transcription.By fluorescence quantitative PCR and Western blots,we examined the effects of CLA4 and CDH1 knockout and Set1-S228A mutation on histone expression levels.Our results showed that Cdh1 down-regulated histone gene transcription by degrading Set1 protein.Cla4increases the stability of Set1 protein by phosphorylating Set1-S228 to promote histone expression.In summary,we found that Cla4 inhibits the ubiquitination of Set1 by APC/C^Cdh1by catalyzing Set1-S228 phosphorylation.The crosstalk between these two post-translational modifications not only regulates Set1 protein levels,but also regulates histone gene expression.We proposed a molecular mechanism that phosphorylation,ubiquitination and methylation interact with each to maintain yeast normal life activities.This model will provide new ideas and clues for studying the indirect regulation of gene expression by other modification enzymes through cell cycle.