Study On Molecular Mechanism Of RBFOX2 In PRC2 Deposit And H3K27me3 Modification Genome-widely

PRC2 is a histone methyltransferase,which can trimethylate K27 of histone H3(H3K27me3), and thus induce repressed chromatin to repress transcription activity. There is still debate on how PRC2 recruitment in mammalian genome, which is a hot area in epigenitic modification. Biochemical studies reveal that PRC2 can promiscuously bind to RNA and genome-wide data reveals that PRC2 prefers to bind the 5' region of mRNAs from modestly expressed protein-coding genes. Basing on interaction of PRC2 and RNA, a model of PRC2 recruiment by Nascent RNA to promoter region in mammalian genome is proposed. RNA binding protein FOX2(RBFXO2) is a splicing factor and widely expressed in many different tissues and cell types. It plays a critical role in nervous tissue?myocardium and muscular tissue development and normal function maintaince. Besides splicing regulation function, a transcription repression role of RBFOX2 is certified in our previous unpublished data. For understanding the transcription repression mechanisim of RBFOX2, we perform a series of experiments and find that RBFOX2 can recruit PRC2 to repress transcription activity, which is regulted by ERK1/2 signal pathway in ESCs. The main study work and results are summarized as follow:(1) RBFOX2 ChIP-seq data shows that RBFOX2 binding sites are enriched in transcription start site(TSS) region, which is a striking global concordance with the binding patterns of SUZ12, a key component of PRC2. And RBFXO2 singal peak is highly overlaped with the signal peak of SUZ12. Further study shows RBFOX2 recruiment to promoter region is dependent on nascent RNA.(2) In vitro co-immunoprecipitation(Co-IP) data shows that RBFOX2 can directly interact with PRC2 independent on RNA or DNA. GST-Pulldown assay reveals that GST-RBFOX2 can capture whole PRC2 dependent on C-terminal domain(CTD).(3) For regular culture condition, RBFox2 knockdown can significantly reduce H3K27me3 ChIP-seq signals on promoter region in ESCs genome-widely, but not “2i”(inhibitor of ERK1/2 PD0325901 and inhibitor of GSK3 CHIR99021) culture condition. Futhermore, “2i” culture condition causes a striking decrease of H3K27me3 on promoter region compared with regular culture condition. However, the reducing level of H3K27me3 with RBFOX2 is significant higher than that without RBFOX2.(4) There are two major isoforms of RBFOX2(RBFOX2a and RBFOX2f) in different cell types. During stem cell differentiation, the major isoform transits from RBFOX2 a to RBFOX2 f. Actived ERK1/2 signal pathway can phosphorylate RBFOX2 a, but not RBFOX2 f.(5) The phosphorylation of RBFOX2 a can not change RBFOX2 a location and interaction with PRC2. However, phosphorylated RBFOX2 a shows a strong RNA binding ability and splicing regulation function.Together with results above, we get a new model for PRC2 recruitment. RBFOX2 may initially interact with nascent RNAs on chromatin and then recruit PRC2 via protein-protein interactions. On highly active genes, actived histone marker H3K4me3 and H3K36me3 will inhibit PRC2 activity. On modestly expressed genes, the recruited PRC2 may catalysis H3K27me3 to repress transcription activity. These findings therefore suggest that nascent RNAs produced from protein-coding genes are not only the products of gene expression, but also critical signals for maintaining homeostatic gene expression. Furthermore,2i culture condition can push ESCs to na?ve state, in which the H3K27me3 midification is significantly reduced genome-widely. ERK1/2 inhibitor(PD0325901) dephosphorylated RBFOX2 to reduce RNA binding ability, thereby to reduce PRC2 recruiment, which is partly responsible for low H3K27me3 in navie ESCs.