The Role Of Polycomblike Proteins In Liquid-Liquid Phase Separation

Polycomb Group(PcG)proteins were first discovered in Drosophila,and Polycomblike Proteins(PCLs)were the second discovered PcG members.They are accessory proteins of core subunit of the PRC2 complex which belongs to histone methyltransferase cofactors.Polycomblikes(PCLs)include PCL1(PHF1),PCL2(MTF2),PCL3(PHF19),hereinafter referred to as PCLs,playing important role in the post-translation of histone modification and gene transcription.However,the specific mechanism of this action is not completely clear.Liquid-liquid phase separation(LLPS),as a new regulatory mechanism,has been fully demonstrated in recent years.LLPS is involved in various biological processes such as transcription,chromatin condensation,and DNA damage.Some diseases originate from the abnormal phase separation of proteins,such as neurodegenerative diseases,cancer and so on.Proteins capable of LLPS usually contain inner disorder region(IDR),acidic or basic protein domains within them.During the previous exploration in our laboratory,it was found that PCLs can exhibit the characteristics of LLPS.And the early mass spectrometry(MS)results showed that PCLs can interact with the nucleolar protein B23/NPM1 which forms phase separation regulation.We verified the interaction between PCLs and NPM1 by coimmunoprecipitation(Co-IP).Subsequent immunofluorescence,high-resolution imaging techniques(advanced applications such as Fluorescence recovery after photobleaching,slice scanning,and time-series imaging)were used to confirm that PCLs form LLPS,and LLPS was found to be associated with nucleoli.We used lentivirus infection to construct stable transfection cell lines and found that LLPS of PCLs was independent of PRC2.At the same time,it was found that nucleoli smeared in PCLs knockdown stable cell lines and in PRC2 core member knockdown stable cell lines,leading to increased apoptosis in cell cycle arrest.This study associates epigenetic regulators with LLPS,and provides new ideas for transcriptional regulators to perform functions through LLPS.Research methods:To clarify the existence of LLPS in PCLs;to clarify the interaction relationship between PCLs and nucleoli through phase separation;to clarify the influence of LLPS involved in PCLs on cell life activities.Using cell lines with high expression of PCLs as the research object,the expression levels of PCLs and the core members of the PRC2 complex,EZH2 and SUZ12,were knocked down or overexpressed by lentivirus infection,and the relationship between PCLs and B23/NPM1.Looking for the biological process in which PCLs play an important role in phase separation.Research methods:1)Using immunofluorescence to detect the localization and distribution characteristics of PCLs and PRC2 complex core subunit EZH2 and SUZ12 in cells2)Verify LLPS of PCLs by high-resolution imaging3)Mass spectrometry shown the interaction protein B23/NPM1 with phase separation,and Co-IP further verified the interaction relationship4)To construct domain truncation mutants to find key domains of PCLs in LLPS5)Construct PCLs and PRC2 complex core members EZH2,SUZ12 knockdown(KD)stably transfected cell lines to observe the changes in the localization relationship between PCLs and NPM16)Detection of PCLs and PRC2 complex core members EZH2,SUZ12 knockdown cell proliferation and apoptosisResearch results:1)PCLs and EZH2,SUZ12 and nucleolus have a common positioning situation2)PCLs have the characteristics of LLPS3)PCLs and nucleolus GC region ingredients NPM1 have an interactive relationship4)LLPS formed by PCLs requires the full-length protein5)The morphology of NPM1 in the PCLs KD cell line changes,showing a diffuse distribution6)PCLs KD cell lines showed cell cycle arrest and increased apoptosisConclusion:PCLs have Liquid-Liquid phase separation,and the LLPS of PCLs depend on the full-length protein to perform normal localization;the LLPS of PCLs is related to the nucleolus protein B23/NPM1,and in PCLs KD and PRC2 core components EZH2,SUZ12.The nucleolus in KD cell lines are abnormal in shape and smear,resulting in cell cycle arrest and increased apoptosis.