T细胞蛋白p80调控c-Myc表达的机理
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摘要
为了解p80在正常T细胞以及T细胞癌变过程中的作用,在缺失p80的T淋巴瘤细胞中重新表达p80,其结果显示:原癌基因c-Myc的表达受到显著抑制,且细胞周期在G1期出现了停滞.定量PCR的结果表明:p80对c-Myc表达的抑制是转录水平的抑制.双荧光素酶报告基因试验表明:p80对c-Myc的抑制作用定位于c-Myc启动子上相对于转录起始位点的-1042bp～-630bp区域.运用TFSEARCH软件对这一区域的分析发现存在多个c-Myb结合位点.在稳定表达p80的T淋巴癌细胞中敲低c-Myb,表明p80对c-Myc的转录抑制是通过c-Myb来实现的.免疫共沉淀实验表明p80和c-Myb在细胞中存在相互作用.进一步的研究显示p80对c-Myc的转录抑制依赖于组蛋白去乙酰化酶的活性.研究结果表明p80有可能通过与c-Myb的相互作用将组蛋白去乙酰化酶募集至c-Myc启动子区域,并通过组蛋白的去乙酰化抑制c-Myc的表达.
Our experimental data indicate that overexpression of p80 in p80 null T cell leukemia cells significantly down-regulates c-Myc oncoprotein and blocks cell cycle progression from G1 to S phase. The qPCR examination and protein stability test found that p80 regulates expression of c-Myc at the transcriptional level. The analysis of c-Myc promoter activity at the presence of p80 by Dual-Luciferase Reporter Assay indicated that the transcriptional repression of p80 on c-Myc promoter is located at-1042 bp～-630 bp above the transcription start site of c-Myc gene. The transcription factor binding sites search of this region by software TFSEARCH revealed multiple binding sequences of c-Myb, another oncoprotein and transcription factor which plays an important role in the proliferation and differentiation of blood cells during hematopoiesis. Knockdown c-Myb in MOLT-4 cells stably expressing p80 indicate that p80 suppresses the transcription of c-Myc gene through c-Myb. Co-immunoprecipitation experiment demonstrated that p80 interacts with c-Myb inside cells. The further experiment has shown that transcriptional repression of c-Myc by p80 is dependent on the enzymatic activity of histone deacetylase. Taken together, our results suggest that p80 recruits histone deacetylase to the c-Myc promoter through interaction with transcription factor c-Myb, and p80 suppresses c-Myc expression by histone deacetylation.
Our experimental data indicate that overexpression of p80 in p80 null T cell leukemia cells significantly down-regulates c-Myc oncoprotein and blocks cell cycle progression from G1 to S phase. The qPCR examination and protein stability test found that p80 regulates expression of c-Myc at the transcriptional level. The analysis of c-Myc promoter activity at the presence of p80 by Dual-Luciferase Reporter Assay indicated that the transcriptional repression of p80 on c-Myc promoter is located at-1042 bp～-630 bp above the transcription start site of c-Myc gene. The transcription factor binding sites search of this region by software TFSEARCH revealed multiple binding sequences of c-Myb, another oncoprotein and transcription factor which plays an important role in the proliferation and differentiation of blood cells during hematopoiesis. Knockdown c-Myb in MOLT-4 cells stably expressing p80 indicate that p80 suppresses the transcription of c-Myc gene through c-Myb. Co-immunoprecipitation experiment demonstrated that p80 interacts with c-Myb inside cells. The further experiment has shown that transcriptional repression of c-Myc by p80 is dependent on the enzymatic activity of histone deacetylase. Taken together, our results suggest that p80 recruits histone deacetylase to the c-Myc promoter through interaction with transcription factor c-Myb, and p80 suppresses c-Myc expression by histone deacetylation.
引文
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[4]Chupp G L,Wright E A,Wu D,et al.Tissue and T cell distribution of precursor and mature IL-16[J].J Immunol,1998,161:3114.
[5]Cristillo A D,Bierer B E.Identification of novel targets of immunosuppressive agents by cDNA-based microarray analysis[J].J Biol Chem,2002,277:4465.
[6]Feske S,Giltnane J,Dolmetsch R,et al.Gene regulation mediated by calcium signals in T lymphocytes[J].Nat Immunol,2001,2:316.
[7]Wilson K C,Center D M,Cruikshank W W,et al.Binding of HTLV-1tax oncoprotein to the precursor of interleukin-16,a T cell PDZ domain-containing protein[J].Virology,2003,306:60.
[8]Ren F,Zhan X,Martens G,et al.Pro-IL-16regulation in activated murine CD4+lymphocytes[J].J Immunol,2005,174:2738.
[9]Zhang Y,Tuzova M,Xiao Z X,et al.Pro-IL-16recruits histone deacetylase 3to the Skp2core promoter through interaction with transcription factor GABP[J].J Immunol,2008,180:402.
[10]Amati B,Alevizopoulos K,Vlach J.Myc and the cell cycle[J].Front Biosci,1998,3:d250.
[11]Henriksson M,Luscher B.Proteins of the Myc network:essential regulators of cell growth and differentiation[J].Adv Cancer Res,1996,68:109.
[12]Nesbit C E,Tersak J M,Prochownik E V.MYConcogenes and human neoplastic disease[J].Oncogene,1999,18:3004.
[13]Westin EH,Gallo RC,Arya SK,et al.Differential expression of the amv gene in human hematopoietic cells[J].Proc Natl Acad Sci U S A,1982,79:2194.
[14]Wasner M,Tschop K,Spiesbach K,et al.Cyclin B1transcription is enhanced by the p300coactivator and regulated during the cell cycle by a CHR-dependent repression mechanism[J].FEBS Lett,2003,536:66.
[15]Wasner M,Haugwitz U,Reinhard W,et al.Three CCAAT-boxes and a single cell cycle genes homology region(CHR)are the major regulating sites for transcription from the human cyclin B2 promoter[J].Gene,2003,312:225.
[16]Spender L C,Inman G J.Developments in Burkitt's lymphoma:novel cooperations in oncogenic MYCsignaling[J].Cancer Manag Res,2014,6:27.
[17]Bretones G,Delgado M D,Leon J.Myc and cell cycle control[J].Biochim Biophys Acta,2015,1849:506.
[18]陈静,易勇,贺寒冰,等.△Np63γ和△Np63a及其C端在H1299细胞凋亡中的作用[J].四川大学学报:自然科学版,2013,50:605.
[19]王承恩,罗玉,贺正池,等.长非编码RNALINC00941在结直肠癌中的表达及对细胞增殖的影响[J].四川大学学报:自然科学版,2017,54:1301.
[20]Biedenkapp H,Borgmeyer U,Sippel A E,et al.Viral myb oncogene encodes a sequence-specific DNA-binding activity[J].Nature,1988,335:835.
[21]Cruikshank W,Little F.lnterleukin-16:the ins and outs of regulating T-cell activation[J].Crit Rev Immunol,2008,28:467.
[22]Center D M,Cruikshank W W,Zhang Y.Nuclear pro-IL-16regulation of T cell proliferation:p27(KIP1)-dependent G0/G1arrest mediated by inhibition of Skp2transcription[J].J Immunol,2004,172:1654.
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[24]Kanei-Ishii C,MacMillan E M,Nomura T,et al.Transactivation and transformation by Myb are negatively regulated by a leucine-zipper structure[J].Proc Natl Acad Sci U S A,1992,89:3088.