PHF20L1 As A Novel H3K27me2 Reader Coordinates With PRC2 And The NuRD Complex To Promote Breast Tumorigenesis

Objectives TUDOR domain-containing proteins(TDRDs)are chiefly responsible for recognizing methyl-lysine/arginine residues.Methyl-lysine/arginine histone recognition by ‘‘reader’’ proteins constitutes a sophisticated gene expression regulatory network involving various biological processes.However,how TDRDs dysregulation contributes to breast cancer development is poorly understood.PHF20L1 were reported to recognize non-histone methylation.However,its roles to recognize histone in breast cancer need further clarification.Several groups have demonstrated that PRC2 and the NuRD complex could synergistically mediate the homeostasis of methylation and acetylation on H3K27 residue.However,the regulation of the modifications on H3K27 site by PRC2 and NuRD remains to be further explored in cancers.Therefore,this study aims to explore following three questions.1.How PHF20L1 dysregulation contributes to breast cancer development;2.The roles of PHF20L1 to recognize histone tails in breast cancer;3.The regulation of the modifications on H3K27 site by PRC2 and NuRD in breast cancer.Methods 1.Growth curve and Ed U assays were performed in MDA-MB-231 cells transfected with si RNAs against the indicated TDRDs to identify new regulators of breast cancer proliferation.2.The histone peptide array was used to screen for potential binding sites of PHF20L1,then peptide pull-down assays,ITC and SPR analysis were performed to verify the recognition between TUDOR and H3K27me2.3.HEK293 T cellular extracts stably expressed FLAG-PHF20L1,were subjected to affinity purification,silver staining to identify PHF20L1 interacting proteins.And co-immunoprecipitation and GST/His pull-down assays were performed.The Gal4 luciferase reporter systems to test the transcriptional activity of PHF20L1.4.Chromatin immunoprecipitation-sequencing(Ch IP-Seq)experiments were performed in normal or PHF20L1-depleted MDA-MB-231 cells as indicated.And q Ch IP analysis on selected genes in the MDA-MB-231 cells after transfection with the sh RNAs against PHF20L1 were performed.5.RNA-sequencing analysis of transcriptional targets for PHF20L1,Gene Set Enrichment Analysis(GSEA)analysis were performed,q Ch IP experiments to detect of MYC and HIF1 A binding sites on the PHF20L1 promoter.Using a Seahorse XFe24 system(Seahorse Bioscience)to measure the glycolysis levels caused by loss-of-function of PHF20L1 or other indicated treatments.6.Propidium iodide FACS analysis,colony formation assays,transwell invasion assays were performed to explore the effect of PHF20L1 on cell cycle,proliferation and metastasis of breast cancer cells.The colony formation assays and transwell invasion assays were performed to evaluate the synergistic effect of PHF20L1,MTA1 and EZH2 on breast cancer cell proliferation and metastasis.Mammary gland tumor formation experiment and tail vein injection cells metastasis models to assess the effect of PHF20L1 in breast cancer process.7.Breast carcinoma samples were collected to analyze the expression profiles by IHC staining,and further data mining to explore the clinicopathological relevance of the MYC/HIF1a-(PHF20L1-MTA1-EZH2)-FOXK2/HIC1 axis in breast cancer.IHC staining of PHF20L1 in multiple carcinomas and Gene expression profiling interactive analysis across all tumor samples and paired normal tissues to explore the expression of PHF20L1 among different cancers.8.Phf20l1 knockout mice were established using CRISPR/Cas9-mediated genome editing to explore the of PHF20L1 in the development of mice.Phf20l1-/-conditional knockout(CKO)mice were generated,by crossed mice bearing floxed Phf20l1 with MMTV-Cre mice to investigate the role of Phf20l1 in the development of the mammary gland.Results 1.si RNA-based screening identifies PHF20L1 as a new regulator of breast cancer proliferation.2.The TUDOR domain of PHF20L1 is a novel H3K27me2-recognizing module.3.PHF20L1 is a transcriptional repressor and interacts with PRC2 and the NuRD complex.4.Colocalization of PHF20L1 with H3K27me2 across the genome is required for PRC2 and NuRD deposition.5.As an MYC and HIF1-driven oncogene,PHF20L1,together with PRC2 and NuRD complex,directly inhibits a series of tumor suppressors to regulate glycolysis.6.PHF20L1 acts in concert with its associated corepressor complexes to promote breast cancer carcinogenesis.7.Clinical relevance of the MYC/HIF1-(PHF20L1-NuRD-MTA1)-FOXK2/HIC1 axis in breast cancer.8.Phf20l1 deletion induces growth retardation and delay of mammary ductal outgrowth in vivo.Conclusion Here,we report that a TUDOR domain containing protein PHF20L1 as a novel H3K27me2 reader exerts transcriptional repression by recruiting polycomb repressive complex 2(PRC2),Mi-2/nucleosome remodeling and deacetylase(NuRD)complex into specific chromatin regions,which links PRC2-mediated methylation and NuRD-mediated deacetylation of H3K27.Furthermore,PHF20L1 was found to serve as an MYC and HIF1-driven potential oncogene,promoting proliferation,metastasis,and glycolysis of breast cancer cells by directly inhibiting tumor suppressors,such as FOXK2 and HIC1.PHF20L1 knockout mice exhibited a severe growth retardation phenotype,and conditional deletion of PHF20L1 contributed to inappropriate mammary gland development.PHF20L1 expression also strongly correlated with higher histologic grades of breast cancer and marked upregulation in various human cancers.Our study suggests that PHF20L1 is a promising target for cancer therapy and exploring the function of PHF20L1 in breast cancer will be beneficial for providing more constructive and effective treatment strategies.