Histone H3K14M Inhibition Of Methyltransferase From Legionrlla Pneumophilia

Part I HISTONE H3K14M INHIBITION OF METHYLTRANSFERASE FROM LEGIONRLLA PNEUMOPHILIAObjective:Legionella pneumophila encodes effector,RomA,a SET domain containing methyltransferase,trimethylates K14 of histone H3 to disturb host gene epigenetic regulation and promote Legionella pneumophila replication and proliferation in host cells.Recently research has shown that,histone lysine-to-methionine(K-to-M)mutations block the methylation of corresponding lysines on wild type histones.Therefore,we try to investigate the effect of histone H3K14 M mutation on the activity of methyltransferase RomA,a secreted effector of Legionella pneumophila in eukaryotic host cells and the underlying mechanisms.Methods:We constructed H3 WT and H3K14M,H3K14I,H3K14 R mutant plasmids,and packaged lentiviral with these plasmids to infect eukaryotic cells such as 293 T and THP-1 with or without over-expression RomA.The H3K14 methylation and acetylation were analyzed by western blotting.The interaction of RomA with H3K14 M was detected by co-immunoprecipitation.Results:A secreted effector of Legionella pneumophila named RomA targeted the host cell nucleus to upregulate the H3K14 methylation level and downregulate the H3K14 acetylation level for inhibiting the gene expression in host cells and promoting Legionella pneumophila's efficient intracellular replication.But histone H3K14 M mutation can promote the interaction between H3K14 and RomA and thus inhibit the methyltransferase activity of RomA.Conclusion:Histone H3K14M mutation significantly inhibits the methyltransferase activity of Legionella pneumophila RomA.Part II TREAT MYC-AMPLIFIED MEDULLOBLASTOMA EFFECTIVELY BY TARGETING EIF4AObjective: Medulloblastoma(MB)is a highly malignant pediatric brain tumor with high morbidity rate,and Group 3(G3)is the most aggressive among four subtypes: WNT,SHH,G3,G4 and without targeted medicine in clinic.MYC amplification or high expression is the typical characteristic of G3 MB and indicates a poor clinical outcome.More importantly,MYC has proven to be an effective drug target for G3 MB,so it's urgently to find a strategy that can effectively target MYC.Methods: We evaluated the RNA and protein stability of MYC,and try to overcome MYC-amplified MB by targeting transcription or translation of MYC.Whereafter,we found that translation-related genes were highly expressed in G3 MB relative to normal brain tissue or other subtypes of MB and highly correlated with MYC expression through R2 database analysis.e IF4A1 inhibition through CRISPR/Cas9 or Silvestrol(e IF4A1i)validated the anti-MYC-amplified MB effect of inhibiting e IF4A1 in vitro and in vivo.Furthermore,the inhibitory mechanisms were investigated via an Ed U proliferation assay,an apoptosis assay,western blotting and cell viability detection.Results: The RNA and protein stability of MYC is low.We observed that the subunit of e IF4 F complex e IF4A1 was highly correlated with MYC in G3 MB and the RNA level of e IF4A1 was higher than normal cerebellum and other subtype MB significantly.The anti-tumour effect of inhibiting e IF4A1 was validated both in vitro and in vivo by CRISPR/Cas9.Moreover,Silvestrol(e IF4A1i)inhibited G3 MB by downregulating the MYC protein level.The exogenous expression of MYC in cells with MYC low expression increased the effect of Silvestrol,and on the opposite,knockdown of MYC in cells with MYC high expression reduced the effect of Silvestrol on cell viability.Conclusion: Our study shows that targeting MYC transcription by e IF4A1 inhibition could be an effective therapeutic strategy against MYC-amplified MB.