Chromatin Microenvironment Promotes PiRNA Transcription By Recruiting The USTC Complex

Since the discovery of RNA interference,the diversity of regulatory small RNAs has been increasing,more and more evidence has revealed that small RNAs play an important role in the regulation of gene expression Piwi-interacting RNAs(piRNA)is a class of small non-coding RNAs identified in animal germ cells.piRNA is highly conserved and is very important for germ cell development in many organisms such as fruit flies,nematode,silkworms,and mice.piRNA and its binding protein Piwi maintain the stability and integrity of genomic DNA in germ cells by silencing transposable elements.At present,the mechanism of piRNA regulating gene expression at the transcription level and post-transcrptional level has been understood,but there are relatively few studies on the upstream transcriptional process.In Caenorhabditis elegans,most piRNA precursors are transcribed from two genomic clusters that contain thousands of individual piRNA transcription units on chromosome ?,and there is a conserved Ruby motif in the promoter regron.As an upstream sequence transcription complex,the USTC complex contains four protein included PRDE-1,TOFU-4,TOFU-5 and SNPC-4.It can recognize the Ruby motif upstream of piRNNA genes and promote the transcription of piRNA precursors through RNAPol ?.USTC complex binds chromosome to locate on the inner nuclear membrane(INM)and forms obvious piRNA focus in the germline nuclei,which indicates the binding and enrichment in the piRNA clusters of chromosome ?.In this work,we used the USTC complex as a tool for large-scale genetic screening.Through genetic,cell biology and molecular biology experimental methods,we identified several histone modification and chromatin remodeling factors that are required for the piRNA focus formation,DNA binding and piRNA biogenesis.In the absence of MES-2,MES-3,MES-6,ISW-1 and MRG-1,the USTC complex is depleted from the piRNA focus and piRNA expression dramatically decreased.Interestingly,MES-2,MES-3 and MES-6 are subunits of the PRC2 complex,which is engaged in histone 3 lysine 27 trimethylation(H3K27me3).The binding of USTC complex in piRNA clusters is related to the modifcation of H3K27me3.Meanwhile,ISW-1 and MRG-1,as chromatin-related proteins,are required for chromatin remodeling and modification.We constructed ISW-1:GFP transgenic nematode in situ using CRISPR/Cas9 technology,found that ISW-1 is widely expressed in somatic and germ cells,and localized in the nucleus.In addition,we also found that the regulation of piRNA by temperature changes is also achieved through altering the binding of USTC complex with piRNA genes to regulate piRNA transcription.High temperature stress can affict the subcellular Iocaization of USTC complex,causing the transcription of the two types of piRNA blocked.Therefore,a proper chromatin microenvironment promotes the transcription of piRNA by recruiting the USTC complex.In general,this work contributes to the understanding of the process of piRNA transcription in C.elegans.And it also has extensive significance for understanding the chromatin microenvironment around the piRNA clusters and how the environmental stimuli regulate piRNA transcription.