Function And Mechanism Of Lysine Methylation On Transcription Factor YY2

Yin Yang 1(YY1)is a multifunctional transcription factor,which can initiate,activate or repress gene transcription.It is vital in various biological processes.YY2 is the paralog of YY1 in mouse and human,and it was initially found through DNA and amino sequence database analysis.DNA and protein amino sequence of human YY2 share 65%and 56%similarity with YY1 respectively.The most notable conserved domain is the zinc finger region.Its function is redundant or opposite compared with YY1.Similar to YY1,YY2 has both transcriptional activation and repression activity.Inhibition of YY2 accelerated cell proliferation and reversed the antiproliferative effects of YY1 deficiency.Despite YY2 possesses important functions,how YY2's functions are regulated,particularly by post-translational modifications(PTMs)remain totally unclear.We previously reported that lysine methyltransferase SET7/9 methylates two conserved lysine sites K173 and K411 of YY1.According to the homology of YY1 and YY2,We hypothesized that YY2 may also be subjected to lysine methylation.In order to identify the lysine methyltransferase of YY2,we screened a list of enzymes in this superfamily which can target histones H3 and H4 for methylation by using in vitro methylation assay,and successfully found that SET7/9 can methylate YY2.Three potential methylation sites,K139,K247 and K369,were found in YY2 according to the conserved motif of SET7/9.Through in vitro methylation assay and in vivo assay following mass spectrum,we determined that K247 can be mono-methylated by SET7/9.LSD1,the first identified lysine demethylase with opposite activity compared to SET7/9 was determined to be the demethylase that can target K247 monomethylation.K247 monomethylation mediated by SET7/9 regulates YY2 DNA binding activity in vitro by EMSA and YY2 interaction with chromatin by ChIP-seq in vivo.Knockout of YY2,SET7/9 and LSD1 by CRISPR(clustered,regularly interspaced,short palindromic repeats)/Cas9-mediated gene editing followed by RNA-seq and bioinformatic analysis revealed that a subset of genes positively regulated by YY2 and SET7/9,but negatively regulated by LSD1,are involved in cell proliferation through GO analysis.Importanly,YY2-regulated gene transcription,cell proliferation and tumor growth are partially dependent on YY2 K247 methylation.Somatic mutations close to K247(K244Q and S246F)found in tumor samples in COSMIC database change YY2 K247 methylation,DNA binding activity and gene transcription YY2 controls,suggesting that YY2 K247 methylation might be clinical relevant.In conclusion,we found the first PTM of YY2,K247 monomethylation,and its regulatory function on YY2 itself.Our results suggested that K247 monomethylation was dynamically regulated by SET7/9 and LSD1,which regulated YY2 DNA binding activity and interaction with chromatin,and therefore YY2-regulated gene transcription,cell proliferation and tumor growth.