The Role Of The Histone Demethylase Rphl In DNA Damage And Regulatory Mechanism Underlying Its Degradation

Histone modifications are well identified as regulators of chromatin structure and transcription,and the enzymes responsible for histone modifications are termed as"writer" of epigenetics.Histone methyltransferases and demethylases catalyze histone methylation and participate in chromotin remodelling,DNA replication,transcription elongation and DNA repair.In Saccharomyces cerevisiae,there exist five JmjC-containing demethylases,including Jhdl,Jhd2,Rph1,Gisl,and Ecm5.In this project,we investigated the role of histone demethylase Rphl in response to DNA damage signaling.We reported that Rphl is a transcriptional repressor of several DNA damage-related genes.Rph1 represses gene expression irrelavant of its demethylase activity.We attempted to identify its targets at genomic locus by Rphl via ChIP-Seq technology,unexpectedly,we only identified some genes required for ribosome synthesis but not for DNA damage response.Moreover,Rph1 interacts with one of the subunits of RNA Polymerase I complex,Rpa190,in vivo,indicating that Rphl may play a role in rRNA transcription.In the DNA damage stress conditions,Rphl was degraded to compromise its repression role in gene expression.Instead of destructed by 26S proteasome proteolysis,Rphl was degraded via the autophagy pathway.Deletion of ATGs genes or inhibition of lysosome activity compromises Rphl turnover.Gcn5 mediates Rph1 acetylation to license its degradation upon DNA damage.Acetylated Rphl was translocated from the nucleus to the cytoplasm,and subsequently delivered into yeast vacuoles,which allows Rphl to be declined.By mass spectromery,we have identified 8 potential lysine acetylation sites of Rphl.However,mutation of all 8 acetylation sites simutenously did not affect Rphl degradation after MMS treatment.Thus,we proposed that Rphl represses multiple gene expression in normal conditions.For the sake of maintaining proper cellular homeostasis upon DNA damage stress,Rphl has to be degraded in a Gcn5-mediated acetylation manner by autophagy.In summary,we here have established a relationship between Rphl and DNA damage repair(DDR)and autophagy pathway.We showed that the degradation of Rph1 is required for appropriate activation of DNA damage response.More importantly,acetylation-mediated degradation through autophagy may be a general mechanism upon DNA damage to facilitate the clearance of the disordered organelle and repressors of DDR.