CBP/P300介导的PTIP乙酰化可能促进下游肿瘤相关基因CCDC121和GPR75的转录
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摘要
PTIP (Pax transactivation domain-interacting protein,PAXIP1)蛋白参与MLL3 (lysine methyltransferase 2C,KMT2C)/MLL4(lysine methyltransferase 2B,KMT2B)组蛋白H3K4甲基转移酶复合体,促进基因的转录激活。但关于PTIP蛋白的乙酰化修饰及人宫颈癌细胞中PTIP转录激活靶基因的研究尚无报道。本研究以稳定表达SFB-PTIP蛋白的293T细胞株为对象,通过免疫沉淀和Western印迹法发现,PTIP蛋白能够发生乙酰化修饰,乙酰转移酶CBP/P300介导PTIP蛋白的乙酰化过程,CBP是主要乙酰转移酶。去乙酰化酶HDAC1/2/3介导PTIP蛋白的去乙酰化过程。选择性HDAC1-3抑制剂MS-275促进PTIP蛋白乙酰化水平上升,且呈剂量和时间依赖性。以人宫颈癌HeLa细胞为研究对象,RNA-seq和RT-qPCR证明,CCDC121 (coiled-coil domain containing121)和G蛋白偶联受体75 (G protein-coupled receptor 75,GPR75)是PTIP的转录激活靶基因(P<0. 01)。和对照相比,MS-275介导的蛋白质乙酰化水平上升,促进基因CCDC121和GPR75的mRNA转录呈现不同程度的剂量依赖性上升(P<0. 01,P<0. 05)。由此推测,MS-275有可能是通过促进PTIP蛋白的乙酰化水平,促进CCDC121和GPR75的转录。但其详细机制有待进一步研究。本研究对今后深入探讨PTIP乙酰化修饰对下游肿瘤相关基因转录的调节和功能,如肿瘤发生、进展等方面的调控机制提供了基础。
PTIP( Pax transactivation domain-interacting protein,PAXIP1) is a component of the histone H3 K4 methyltransferase complex that also contains MLL3( lysine methyltransferase 2 C,KMT2 C)or MLL4( lysine methyltransferase 2 B,KMT2 B) to activate gene transcription. However,there is no report on PTIP acetylation or its transcription-activated target genes in He La cells. In this study,we used immunoprecipitation and Western blotting assays in 293 T cells that stably expressed protein SFB-PTIP,and found that CBP/P300 were acetyltransferases that modulated PTIP acetylation. Moreover,HDAC1/2/3 mediated the deacetylation of PTIP,and the selective HDAC1-3 inhibitor MS-275 increased the acetylation of PTIP in a dose-and time-dependent manner. In addition, RNA-seq and RT-qPCR identified that CCDC121( coiled-coil domain-containing 121) and GPR75( G protein-coupled receptor75) were PTIP-activated target genes( P < 0. 01) in HeLa cells. Compared with the control,MS-275-mediated increase of protein acetylation up-regulated CCDC121 and GPR75 mRNA transcription levels in a dose-dependent manner( P<0. 01,P<0. 05). We supposed that MS-275 may promote the transcription of CCDC121 and GPR75 by increasing PTIP acetylation,but the mechanism is still unclear. This study provided a basis for further inquiry into the transcriptional regulation and functions of downstream tumorassociated genes by PTIP acetylation,such as tumorigenesis and progression.
PTIP( Pax transactivation domain-interacting protein,PAXIP1) is a component of the histone H3 K4 methyltransferase complex that also contains MLL3( lysine methyltransferase 2 C,KMT2 C)or MLL4( lysine methyltransferase 2 B,KMT2 B) to activate gene transcription. However,there is no report on PTIP acetylation or its transcription-activated target genes in He La cells. In this study,we used immunoprecipitation and Western blotting assays in 293 T cells that stably expressed protein SFB-PTIP,and found that CBP/P300 were acetyltransferases that modulated PTIP acetylation. Moreover,HDAC1/2/3 mediated the deacetylation of PTIP,and the selective HDAC1-3 inhibitor MS-275 increased the acetylation of PTIP in a dose-and time-dependent manner. In addition, RNA-seq and RT-qPCR identified that CCDC121( coiled-coil domain-containing 121) and GPR75( G protein-coupled receptor75) were PTIP-activated target genes( P < 0. 01) in HeLa cells. Compared with the control,MS-275-mediated increase of protein acetylation up-regulated CCDC121 and GPR75 mRNA transcription levels in a dose-dependent manner( P<0. 01,P<0. 05). We supposed that MS-275 may promote the transcription of CCDC121 and GPR75 by increasing PTIP acetylation,but the mechanism is still unclear. This study provided a basis for further inquiry into the transcriptional regulation and functions of downstream tumorassociated genes by PTIP acetylation,such as tumorigenesis and progression.
引文
[1] Lechner MS,Levitan I,Dressler GR. PTIP,a novel BRCT domain-containing protein interacts with Pax2 and is associated with active chromatin[J]. Nucleic Acids Res,2000,28(14):2741-2751
[2] Cho EA,Prindle MJ,Dressler GR. BRCT domain-containing protein PTIP is essential for progression through mitosis[J]. Mol Cell Biol,2003,23(5):1666-1673
[3] Munoz IM,Jowsey PA,Toth R,et al. Phospho-epitope binding by the BRCT domains of hPTIP controls multiple aspects of the cellular response to DNA damage[J]. Nucleic Acids Res,2007,35(16):5312-5322
[4] Manke IA,Lowery DM,Nguyen A,et al. BRCT repeats as phosphopeptide-binding modules involved in protein targeting[J].Science,2003,302(5645):636-639
[5] Cho YW,Hong T,Hong S,et al. PTIP associates with MLL3-and MLL4-containing histone H3 lysine 4 methyltransferase complex[J]. J Biol Chem,2007,282(28):20395-20406
[6] Munoz IM,Rouse J. Control of histone methylation and genome stability by PTIP[J]. EMBO Rep,2009,10(3):239-245
[7] Froimchuk E, Jang Y, Ge K. Histone H3 lysine 4methyltransferase KMT2D[J]. Gene,2017,627:337-342
[8] Kouzarides T. Chromatin modifications and their function[J].Cell,2007,128(4):693-705
[9] Patel SR,Kim D,Levitan I,et al. The BRCT-domain containing protein PTIP links PAX2 to a histone H3, lysine 4methyltransferase complex[J]. Dev Cell,2007,13(4):580-592
[10] Abraham S,Paknikar R,Bhumbra S,et al. The Grouchoassociated phosphatase PPM1B displaces Pax transactivation domain interacting protein(PTIP)to switch the transcription factor Pax2 from a transcriptional activator to a repressor[J]. J Biol Chem,2015,290(11):7185-7194
[11] Starnes LM, Su D, Pikkupeura LM, et al. A PTIP-PA1subcomplex promotes transcription for Ig H class switching independently from the associated MLL3/MLL4 methyltransferase complex[J]. Genes Dev,2016,30(2):149-163
[12] Baas R,van Teeffelen HAAM,Tjalsma SJD,et al. The mixed lineage leukemia 4(MLL4)methyltransferase complex is involved in transforming growth factor beta(TGF-β)-activated gene transcription[J]. Transcription,2018,9(2):67-74
[13]吕斌娜,梁文星.蛋白质乙酰化修饰研究进展[J].生物技术通报(LüBN,Liang WX. The research progress of protein acetylation[J]. Biotechnol Bull),2015,31(4):166-174
[14] Choudhary C,Kumar C,Gnad F,et al. Lysine acetylation targets protein complexes and co-regulates major cellular functions[J].Science,2009,325(5942):834-840
[15] Huang W,Wang Z,Lei QY. Acetylation control of metabolic enzymes in cancer:an updated version[J]. Acta Biochim Biophys Sin(Shanghai),2014,46(3):204-213
[16] Goodman RH, Smolik S. CBP/p300 in cell growth,transformation,and development[J]. Genes Dev, 2000, 14(13):1553-1577
[17] Glozak MA,Sengupta N,Zhang X, et al. Acetylation and deacetylation of non-histone proteins[J]. Gene,2005,363:15-23
[18] Reed SM, Quelle DE. p53 Acetylation:regulation and consequences[J]. Cancers(Basel),2014,7(1):30-69
[19] Zhang J,Huang JY,Chen YN,et al. Whole genome and transcriptome sequencing of matched primary and peritoneal metastatic gastric carcinoma[J]. Sci Rep,2015,5:13750
[20] Dedoni S,Campbell LA,Harvey BK,et al. The orphan Gprotein-coupled receptor 75 signaling is activated by the chemokine CCL5[J]. J Neurochem,2018,146(5):526-539
[21] Fan F,Roman RJ. GPR75 Identified as the First 20-HETE receptor:a chemokine receptor adopted by a new family[J]. Circ Res,2017,120(11):1696-1698
[22] Garcia V,Gilani A,Shkolnik B,et al. 20-HETE signals through G-protein-coupled receptor GPR75(Gq)to affect vascular function and trigger hypertension[J]. Circ Res,2017,120(11):1776-1788
[2] Cho EA,Prindle MJ,Dressler GR. BRCT domain-containing protein PTIP is essential for progression through mitosis[J]. Mol Cell Biol,2003,23(5):1666-1673
[3] Munoz IM,Jowsey PA,Toth R,et al. Phospho-epitope binding by the BRCT domains of hPTIP controls multiple aspects of the cellular response to DNA damage[J]. Nucleic Acids Res,2007,35(16):5312-5322
[4] Manke IA,Lowery DM,Nguyen A,et al. BRCT repeats as phosphopeptide-binding modules involved in protein targeting[J].Science,2003,302(5645):636-639
[5] Cho YW,Hong T,Hong S,et al. PTIP associates with MLL3-and MLL4-containing histone H3 lysine 4 methyltransferase complex[J]. J Biol Chem,2007,282(28):20395-20406
[6] Munoz IM,Rouse J. Control of histone methylation and genome stability by PTIP[J]. EMBO Rep,2009,10(3):239-245
[7] Froimchuk E, Jang Y, Ge K. Histone H3 lysine 4methyltransferase KMT2D[J]. Gene,2017,627:337-342
[8] Kouzarides T. Chromatin modifications and their function[J].Cell,2007,128(4):693-705
[9] Patel SR,Kim D,Levitan I,et al. The BRCT-domain containing protein PTIP links PAX2 to a histone H3, lysine 4methyltransferase complex[J]. Dev Cell,2007,13(4):580-592
[10] Abraham S,Paknikar R,Bhumbra S,et al. The Grouchoassociated phosphatase PPM1B displaces Pax transactivation domain interacting protein(PTIP)to switch the transcription factor Pax2 from a transcriptional activator to a repressor[J]. J Biol Chem,2015,290(11):7185-7194
[11] Starnes LM, Su D, Pikkupeura LM, et al. A PTIP-PA1subcomplex promotes transcription for Ig H class switching independently from the associated MLL3/MLL4 methyltransferase complex[J]. Genes Dev,2016,30(2):149-163
[12] Baas R,van Teeffelen HAAM,Tjalsma SJD,et al. The mixed lineage leukemia 4(MLL4)methyltransferase complex is involved in transforming growth factor beta(TGF-β)-activated gene transcription[J]. Transcription,2018,9(2):67-74
[13]吕斌娜,梁文星.蛋白质乙酰化修饰研究进展[J].生物技术通报(LüBN,Liang WX. The research progress of protein acetylation[J]. Biotechnol Bull),2015,31(4):166-174
[14] Choudhary C,Kumar C,Gnad F,et al. Lysine acetylation targets protein complexes and co-regulates major cellular functions[J].Science,2009,325(5942):834-840
[15] Huang W,Wang Z,Lei QY. Acetylation control of metabolic enzymes in cancer:an updated version[J]. Acta Biochim Biophys Sin(Shanghai),2014,46(3):204-213
[16] Goodman RH, Smolik S. CBP/p300 in cell growth,transformation,and development[J]. Genes Dev, 2000, 14(13):1553-1577
[17] Glozak MA,Sengupta N,Zhang X, et al. Acetylation and deacetylation of non-histone proteins[J]. Gene,2005,363:15-23
[18] Reed SM, Quelle DE. p53 Acetylation:regulation and consequences[J]. Cancers(Basel),2014,7(1):30-69
[19] Zhang J,Huang JY,Chen YN,et al. Whole genome and transcriptome sequencing of matched primary and peritoneal metastatic gastric carcinoma[J]. Sci Rep,2015,5:13750
[20] Dedoni S,Campbell LA,Harvey BK,et al. The orphan Gprotein-coupled receptor 75 signaling is activated by the chemokine CCL5[J]. J Neurochem,2018,146(5):526-539
[21] Fan F,Roman RJ. GPR75 Identified as the First 20-HETE receptor:a chemokine receptor adopted by a new family[J]. Circ Res,2017,120(11):1696-1698
[22] Garcia V,Gilani A,Shkolnik B,et al. 20-HETE signals through G-protein-coupled receptor GPR75(Gq)to affect vascular function and trigger hypertension[J]. Circ Res,2017,120(11):1776-1788