PHF1 Is A Novel Reader For Histone Arginine Methylation And Coordinates With PRMT5/CRL4B Complex To Promote Tumorigenesis And Metastasis

Histone post–translational modifications regulate chromatin structure and function largely through interactions with effector proteins that often contain multiple histone-binding domains.The effector proteins are called readers.More and more histone binding domains are recognized these years just like PHD finger domain,TUDOR domain,WD40 domain,Chromo domain and so on.Such recognition mechanisms confer coordinated regulatory functions in a plethora of chromatin template-based biological processes including gene regulation,DNA replication,and recombination.PHF1 [plant homeodomain(PHD)finger protein 1],which contains linked two kinds of histone reader modules,a Tudor domain and two PHD fingers,is an essential factor for epigenetic regulation and genome maintenance.It has been reported that PHF1 interacts with PRC2 complex and effects the enzyme activity of EZH2 which influence the methylation of H3K27 and transcriptional regulation of HOX genes.Moreover,the TUDOR domain of PHF1 could recognize H3K36me3 and H3tK27me3 which supports the reader function.While significant progress has been made characterizing the function of the Tudor domain,the roles of the two PHD fingers are poorly defined.Here,we demonstrate that the N terminal-PHD finger of PHF1 recognizes symmetric dimethylated H4R3(H4R3me2s)catalyzed by PRMT5-WDR77.However,the C terminal PHD finger of PHF1,instead of binding modified histone,directly interacts with DDB1,the main component of the CUL4B-Ring E3 ligase complex(CRL4B)which is responsible for H2AK119 mono-ubiquitination(H2AK119ub1).We showed that PHF1,PRMT5-WDR77 and CRL4 B reciprocally interact with one another and collaborate as a functional coordinated unit,providing the ‘H4R3me2s-H2AK119ub1' readout of a defined combinatorial transcriptional repressive epigenetic signature.Genome-wide analysis of PHF1/PRMT5/CUL4 B targets identified a cohort of genes including E-cadherin and FBXW7 which are critically involved in cell growth and migration.We demonstrate that PHF1 promotes cell proliferation,invasion and tumorigenesis in vivo and in vitro and found that its expression is markedly upregulated in various human cancers.To better understand the mechanistic role of PHF1,we employed affinity purification and mass spectrometry to identify the proteins that are associated with PHF1.In these experiments,FLAG-tagged PHF1(FLAG-PHF1)was stably expressed in HEK 293 T cells.Co-IP assays were performed to verify the interaction between PHF1,PRMT5 and CRL4 B which remind us the three may act as an organic whole.GST pulldown assays were performed to explore the molecular basis for the interaction.Next,we found PHF1 recognized H4R3me2 s through its PHD finger domain by GST pulldown and peptide pulldown assays.Next,ChIP-on-chip assays were performed to find out the target genes that transcriptional regulated by PHF1/PRMT5/CUL4 B complex.We proved that PHF1,PRMT5-H4R3me2 s and CUL4B-H2AK119ub1 colocalized on the promotors of E-cadherin and FBXW7 and any of them was essential for the complex.The role of PHF1 in tumor progression is still unclear.We found that PHF1 promotes tumor proliferation by growth curve,colony formation and EDU assays.The expression of EMT markers and transwell assays showed that PHF1 promotes invasion of breast cancer.Moreover,cancer cells orthotopically implanted or intravenously injected into NOD-SCID mice to measure orthotopical tumorigenesis or seeding lung metastasis showed that PHF1 promotes tumorigenesis and metastasis of cancer in vivo.We also found that the expression of PHF1 is upregulated in various cancers and associated with poor prognosis.To sum up,our data identified a new reader for H3R4me2 and provide a molecular basis for the functional interplay between histone arginine methylation and ubiquitination.The results also indicate that PHF1 is a key factor in cancer progression,supporting the pursuit of PHF1 as a target for cancer therapy.