The Molecular Mechanism Of PHF6 Regulating NSD2-mediated H3K36me2 Modification

The epigenetic mechanisms that regulate gene expression mainly include DNA methylation,histone modification,and non-coding RNA,among which histone modification plays a crucial role in chromatin regulation.As a marker of transcription activity,histone H3 lysine 36 dimethylation(H3K36me2)was positively correlated with gene transcription level.To date,seven histone methyltransferases have been found to catalyze the dimethylation of H3K36,including NSD1,NSD2,NSD3,ASH1 L,SETD3,SETMAR,and Smy D2.Oncogene nuclear receptor binding SET domain protein 2(NSD2)was first identified in multiple myeloma and is highly expressed in solid tumors including breast,lung,bladder,colon,and prostate cancers.NSD2 is involved in many cellular processes,including DNA damage repair,DNA replication,epithelial-mesenchymal transformation,and cell cycle regulation.As a member of the histone methyltransferase family,NSD2 mainly catalyzes H3K36me2.However,the mechanism by which NSD2 regulates the chromatin distribution of H3K36me2 has been rarely studied.In order to explore the specific mechanism by which NSD2 regulates H3K36me2,we obtained a series of NSD2-bound proteins through immunoprecipitation experiment and mass spectrometry analysis,and selected PHF6 as the candidate protein through protein domain analysis.Through the in vivo immunoprecipitation experiment,we found that PHF6 protein could indeed bind to NSD2,and the deletion of its C-terminal domain led to the reduction of this binding.To further verify the binding effect of PHF6 and NSD2,we conducted in vitro pull-down experiment and found that the C terminal of PHF6 protein was mainly responsible for direct binding with NSD2 protein,while the fourth PHD4 region of NSD2 protein was mainly responsible for direct binding with PHF6 protein.The knockout of PHF6 gene in mouse embryonic stem cells resulted in a decrease in NSD2 protein level and a significant decrease in H3K36me2 and an increase in H3K27me3 level.By alkaline phosphatase staining and MTS cell proliferation assay,we found that PHF6 knockout did not affect the pluripotency and growth rate of mouse embryonic stem cells.In conclusion,the C-terminal of PHF6 regulates NSD2-mediated H3K36me2 by binding to the fourth PHD4 region of NSD2.This study provides a new molecular mechanism for the regulation of H3K36me2 in vivo,and provides more possible targets for the future regulation of intracellular H3K36me2 level.