The Silencing Regulator MOM1 Interacts With The SUMO E3 Ligase-like Proteins PIAL1,PIAL2 And Forms A Complex Required For Transcriptional Silencing In Arabidopsis

In eukaryotes,transposable elements,DNA repeats and exogenous transgenes are usually subjected to transcriptional silencing to keep genome stability.In Arabidopsis thaliana,transcriptional gene silencing is accompanied by DNA methylation and repressive histone marks.But MOM1 was characterized as an important transcriptional silencing regulator independently of changes in DNA methylation,in 2000.The CMM2(conserved MOM1 motif 2)domain of MOM1 was previously shown to form a dimmer in vitro and the C terminal of MOM1 could interact with SUMO(small ubiquitin-like modifier)by yeast two-hybrid analysis.SUMO is a posttranslational modification which can be attached to substrate proteins by SUMO E3 ligases and involved in many biological processes,including DNA replication,transcription,and chromatin remodeling.At present,the mechanism by which MOM1 mediates transcriptional silencing still remains elusive.To identify novel silencing regulators,we generated a luciferase reporter system driven by hypermethylated FWA promoter.By forward genetic screening and map-based cloning,we identified some RdDM(RNA-directed DNA methylation)components and other known silencing regulators.Based on whole-genome sequencing,we also identified a novel silencing regulator,protein inhibitor of activated stat like 2(PIAL2),which is a SUMO E3 ligase-like protein.Based on RNA deep sequencing and whole-genome bisulfite sequencing analyses,we found that PIAL2 and its paralog PIAL1 function redundantly in transcriptional silencing and share common targets with MOM1 independently of changes in DNA methylation.By pull-down in combination with mass spectrometric analysis,coimmunoprecipitation,and gel filtration,we proved that PIAL1,PIAL2,MOM1 can formhomodimers and interact with each other to form a high molecular mass complex in vivo.In absence of either PIAL1/2 or MOM1,the high molecular mass complex is disrupted.We identified a previously uncharacterized IND(interacting domain)in PIAL1 and PIAL2 and demonstrated that the IND domain is involved in transcriptional silencing exclusively through the interaction with MOM1.We also demonstrated that MOM1 can form a homodimer through the CMM2 domain in vivo and the dimerization is required for MOM 1's function in transcriptional silencing.Besides,the CMM2 domain is also required for the interaction of MOM 1 with PIAL1 and PIAL2.By an in vitro sumoylation assay,we confirmed that PIAL2 has SUMO E3 ligase activity and the RING and SIM(SUMO interaction motif)are required for the SUMO E3 ligase activity.However,the SUMO E3 ligase activity is dispensable for PIAL2's function in transcriptional silencing.We demonstrated that,although the CMM3 domain of MOM1 is responsible for the interaction of MOM1 with SUMO,the CMM3 domain is not responsible for MOM1's function in transcriptional silencing.Besides,we did not detect the interaction of MOM 1 with SUMO covalently or noncovalently and demonstrated that the CMM3 domain is dispensable for MOM1's function in transcriptional silencing.Further studies are required to understand whether and how MOM1 is related to SUMO in vivo.Collectively,by using genetic analysis in combination with biochemical and molecular biological methods,we identified two SUMO E3 ligase-like proteins,PIAL1 and PIAL2,and demonstrated that they act as components of the MOM 1-containing complex and mediate transcriptional silencing at heterochromatin regions.While their SUMO ligase activity is not required for transcriptional silencing,the integrity of MOM1-PIAL1/2 complex is required for transcriptional silencing.This study enhances our understanding of how SUMO E3 ligases function in transcriptional silencing and promotes to solve the puzzle of the field——how MOM1 contributes to transcriptional silencing independently of DNA methylation.