A Novel Histone H4Binding Activity And Transcriptional Regulatory Function For Signal Recognition Particle Subunits SRP68and SRP72

The nucleosome is the fundamental unit of chromatin and it is composed of an octamer of the four core histones (H3, H4, H2A, H2B) around which147base pairs of DNA are wrapped. Arginine methylation, which plays a important role on transcription regulation, broadly occurs in the N-teminal tails of core histones. Histone arginine methylation, described as a part of "Histone code", is required for the downstream effctor recruiment or rejection. However, the mechanisms by which histone arginine methylation regulates transcription remain poorly understood. In this study we have attempted to purify nuclear proteins in mammalian cell that recognize specifically the symmetric dimethylated arginine3in histone H4tail(H4R3me2s), the asymmetric dimethylated arginine3in histone H4tail(H4R3me2as) and unmethylated arginine3in histone H4tail using Pull down assay. No major nuclear protein was observed to bind specifically the methylated H4R3peptides. However, both H4R3me2s and H4R3me2as markedly inhibit the binding of two proteins to unmethylated H4R3tail. These proteins were identified as the signal recognition particle SRP68and SRP72heterodimers by an unbiased proteomic approach.SRP68and SRP72are two polypeptides of the signal recognition particle. SRP68and SRP72together with SRP RNA、SRP9、SRP14、SRP19、SRP54could form a SRP complex, which plays an key role in the delivery of secretory proteins and membrane proteins to endoplasmic reticulum membrane.Here we show that only SRP68and SRP72, but not the SRP complex, bind to the unmethylated H4R3peptide according to gel filtration chromatography and pull down assay result. In vitro, SRP68and SRP72bind to H4R3tail in a methylation sensitive manner. In vivo, SRP68and SRP72associate with chromatin and bind to H4N-terminal polypeptide. Chromatin immunoprecipitation and western blot result prove that SRP68/SRP72mainly combine with chromatin without H4R3methylation modification. Overexpressing arginine methylation enzyme PRMT5, also can increase histone arginine symmetric methylation level in the cells, leading to the interaction between SRP68/SRP72protein and chromatin reduce. In the meanwile, SRP68and SRP72cytoplasm localization increase.The genome-wide occupancy study of SRP68reveals a broad distribution of SRP68in genome. The SRP68binding sites were mapped to the known transcription factor motif data, we discovery that the two DNA binding motifs of the transcription factors NFAT and KLF4are dramaticly rich in SRP68binding sites.Together our study underscores a role of H4R3methylation in blocking the binding of effector protein SRP68and SRP72to chromatin. In addition, our study reveals a novel role for SRP68and SRP72in transcriptional regulation.