组蛋白H3K14M突变对嗜肺军团菌来源甲基转移酶活性的抑制
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摘要
目的·探讨真核细胞中组蛋白H3K14M突变对嗜肺军团菌分泌的甲基转移酶RomA活性的影响及其内在机制。方法·构建组蛋白H3野生型(H3WT)以及组蛋白H3上14位赖氨酸突变为甲硫氨酸、异亮氨酸、精氨酸(H3K14M、H3K14I、H3K14R)的真核表达质粒,慢病毒包装并感染稳定表达RomA的293T、THP-1等细胞。用Western blotting检测并比较带有不同H3K14突变的细胞中内源H3K14位点表观修饰的改变。并利用免疫共沉淀技术检测H3K14M与RomA蛋白的相互作用。结果·嗜肺军团菌的分泌效应物RomA通过靶向宿主细胞核,增加其H3K14甲基化水平同时降低H3K14乙酰化水平,从而抑制宿主基因的表达,促进嗜肺军团菌有效的细胞内复制;但组蛋白H3K14M突变可以通过增强与RomA蛋白的相互作用以抑制其甲基转移酶活性,阻断RomA对H3K14位点的甲基化。结论·组蛋白H3K14M突变显著抑制嗜肺军团菌来源甲基转移酶RomA活性。
Objective · To investigate the effect of histone H3K14M mutation on the activity of methyltransferase RomA, a secreted effector of Legionella pneumophila in eukaryotic cells and the underlying mechanisms. Methods · Wide-type histone H3(H3WT) and mutant histone H3(the lysine residue 14 was replaced by methionine, isoleucine or arginine residue, and named as H3K14M, H3K14I, and H3K14R, respectively) recombinant expression plasmids were constructed. Packaged lentiviruses with these plasmids were used to infect eukaryotic cells 293 T and THP-1 with or without over-expression of RomA. The H3K14 methylation and acetylation were analyzed by Western blotting. The interaction of RomA with H3WT and H3K14 mutants 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 H3K14M mutation could promote the interaction between H3K14M and RomA and thus inhibited the methyltransferase activity of RomA. Conclusion · Histone H3K14M mutation significantly inhibits the activity of Legionella pneumophila methyltransferase RomA.
Objective · To investigate the effect of histone H3K14M mutation on the activity of methyltransferase RomA, a secreted effector of Legionella pneumophila in eukaryotic cells and the underlying mechanisms. Methods · Wide-type histone H3(H3WT) and mutant histone H3(the lysine residue 14 was replaced by methionine, isoleucine or arginine residue, and named as H3K14M, H3K14I, and H3K14R, respectively) recombinant expression plasmids were constructed. Packaged lentiviruses with these plasmids were used to infect eukaryotic cells 293 T and THP-1 with or without over-expression of RomA. The H3K14 methylation and acetylation were analyzed by Western blotting. The interaction of RomA with H3WT and H3K14 mutants 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 H3K14M mutation could promote the interaction between H3K14M and RomA and thus inhibited the methyltransferase activity of RomA. Conclusion · Histone H3K14M mutation significantly inhibits the activity of Legionella pneumophila methyltransferase RomA.
引文
[1]Strahl BD,Allis CD.The language of covalent histone modifications[J].Nature,2000,403(6765):41-45.
[2]Lee JS,Smith ER,Shilatifard A,et al.The language of histone crosstalk[J].Cell,2010,142(5):682-685.
[3]Greer EL,Shi Y.Histone methylation:a dynamic mark in health,disease and inheritance[J].Nat Rev Genet,2012,13(5):343-357.
[4]Herz HM,Garruss AS,Shilatifard A,et al.SET for life:biochemical activities and biological functions of SET domain-containing proteins[J].Trends Biochem Sci,2013,38(12):621-639.
[5]Jenuwein T,Laible G,Dorn R,et al.SET domain proteins modulate chromatin domains in eu-and heterochromatin[J].Cell Mol Life Sci,1998,54(1):80-93.
[6]Qian C,Zhou M.SET domain protein lysine methyltransferases:structure,specificity and catalysis[J].Cell Mol Life Sci,2006,63(23):2755-2763.
[7]Stephens RS,Kalman S,Lammel CJ,et al.Genome sequence of an obligate intracellular pathogen of humans:Chlamydia trachomatis[J].Science,1998,282(5389):754-759.
[8]Aravind L,Iyer L M.Provenance of SET-domain histone methyltransferases through duplication of a simple structural unit[J].Cell Cycle,2003,2(4):369-376.
[9]Alvarezvenegas R.Bacterial SET domain proteins and their role in eukaryotic chromatin modification[J].Front Genet,2014:65-65.
[10]Aravind L,Abhiman S,Iyer LM,et al.Natural history of the eukaryotic chromatin protein methylation system[J].Prog Mol Biol Transl Sci,2011:105-176.
[11]Khodr A,Kay E,Gomezvalero L,et al.Molecular epidemiology,phylogeny and evolution of Legionella[J].Infect Genet Evol,2016:108-122.
[12]de Felipe KS,Glover RT,Charpentier X,et al.Legionella eukaryotic-like typeⅣsubstrates interfere with organelle trafficking[J].PLoS Pathog,2008,4(8):e1000117.
[13]Rolando M,Sanulli S,Rusniok C,et al.Legionella pneumophila effector RomAuniquely modifies host chromatin to repress gene expression and promote intracellular bacterial replication[J].Cell Host Microbe,2013,13(4):395-405.
[14]Shan C,Wang J,Xu K,et al.A histone H3K9M mutation traps histone methyltransferase Clr4 to prevent heterochromatin spreading[J].eLife,2016.DOI:10.7554/eLife.17903.
[15]Justin N,Zhang Y,Tarricone C,et al.Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2[J].Nat Commun,2016,7(1):11316.
[16]Lu C,Jain SU,Hoelper D,et al.Histone H3K36 mutations promote sarcomagenesis through altered histone methylation landscape[J].Science,2016,352(6287):844-849.
[17]Bender S,Tang Y,Lindroth AM,et al.Reduced H3K27me3 and DNAhypomethylation are major drivers of gene expression in K27M mutant pediatric high-grade gliomas[J].Cancer Cell,2013,24(5):660-672.
[18]Zhang Y,Reinberg D.Transcription regulation by histone methylation:interplay between different covalent modifications of the core histone tails[J].Genes Dev,2001,15(18):2343-2360.
[19]Zhao B,Xu W,Rong B,et al.H3K14me3 genomic distributions and its regulation by KDM4 family demethylases[J].Cell Res,2018,28(11):1118-1120.
[20]Garcia BA,Hake SB,Diaz RL,et al.Organismal differences in posttranslational modifications in histones H3 and H4[J].J Biol Chem,2007,282(10):7641-7655.
[2]Lee JS,Smith ER,Shilatifard A,et al.The language of histone crosstalk[J].Cell,2010,142(5):682-685.
[3]Greer EL,Shi Y.Histone methylation:a dynamic mark in health,disease and inheritance[J].Nat Rev Genet,2012,13(5):343-357.
[4]Herz HM,Garruss AS,Shilatifard A,et al.SET for life:biochemical activities and biological functions of SET domain-containing proteins[J].Trends Biochem Sci,2013,38(12):621-639.
[5]Jenuwein T,Laible G,Dorn R,et al.SET domain proteins modulate chromatin domains in eu-and heterochromatin[J].Cell Mol Life Sci,1998,54(1):80-93.
[6]Qian C,Zhou M.SET domain protein lysine methyltransferases:structure,specificity and catalysis[J].Cell Mol Life Sci,2006,63(23):2755-2763.
[7]Stephens RS,Kalman S,Lammel CJ,et al.Genome sequence of an obligate intracellular pathogen of humans:Chlamydia trachomatis[J].Science,1998,282(5389):754-759.
[8]Aravind L,Iyer L M.Provenance of SET-domain histone methyltransferases through duplication of a simple structural unit[J].Cell Cycle,2003,2(4):369-376.
[9]Alvarezvenegas R.Bacterial SET domain proteins and their role in eukaryotic chromatin modification[J].Front Genet,2014:65-65.
[10]Aravind L,Abhiman S,Iyer LM,et al.Natural history of the eukaryotic chromatin protein methylation system[J].Prog Mol Biol Transl Sci,2011:105-176.
[11]Khodr A,Kay E,Gomezvalero L,et al.Molecular epidemiology,phylogeny and evolution of Legionella[J].Infect Genet Evol,2016:108-122.
[12]de Felipe KS,Glover RT,Charpentier X,et al.Legionella eukaryotic-like typeⅣsubstrates interfere with organelle trafficking[J].PLoS Pathog,2008,4(8):e1000117.
[13]Rolando M,Sanulli S,Rusniok C,et al.Legionella pneumophila effector RomAuniquely modifies host chromatin to repress gene expression and promote intracellular bacterial replication[J].Cell Host Microbe,2013,13(4):395-405.
[14]Shan C,Wang J,Xu K,et al.A histone H3K9M mutation traps histone methyltransferase Clr4 to prevent heterochromatin spreading[J].eLife,2016.DOI:10.7554/eLife.17903.
[15]Justin N,Zhang Y,Tarricone C,et al.Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2[J].Nat Commun,2016,7(1):11316.
[16]Lu C,Jain SU,Hoelper D,et al.Histone H3K36 mutations promote sarcomagenesis through altered histone methylation landscape[J].Science,2016,352(6287):844-849.
[17]Bender S,Tang Y,Lindroth AM,et al.Reduced H3K27me3 and DNAhypomethylation are major drivers of gene expression in K27M mutant pediatric high-grade gliomas[J].Cancer Cell,2013,24(5):660-672.
[18]Zhang Y,Reinberg D.Transcription regulation by histone methylation:interplay between different covalent modifications of the core histone tails[J].Genes Dev,2001,15(18):2343-2360.
[19]Zhao B,Xu W,Rong B,et al.H3K14me3 genomic distributions and its regulation by KDM4 family demethylases[J].Cell Res,2018,28(11):1118-1120.
[20]Garcia BA,Hake SB,Diaz RL,et al.Organismal differences in posttranslational modifications in histones H3 and H4[J].J Biol Chem,2007,282(10):7641-7655.