ACETYLATION OF LYSINE 564 ADJACENT TO THE CTBP-BINDING MOTIF IN EVI1 IS CRUCIAL FOR TRANSCRIPTIONAL ACTIVATION OF GATA2
ACETYLATION OF LYSINE 564 ADJACENT TO THE CTBP-BINDING MOTIF IN EVI1 IS CRUCIAL FOR TRANSCRIPTIONAL ACTIVATION OF GATA2
复制标题
EVI1 中与 CTBP 结合基序相邻的赖氨酸 564 的乙酰化对于 GATA2 的转录激活至关重要
DOI：
--
复制
发表时间：
2010
期刊：
影响因子：
--
通讯作者：
K. Morishita
中科院分区：
文献类型：
--
作者：
A. Shimahara;N. Yamakawa;I. Nishikata;K. Morishita
转染到COS7细胞中，通过抗P/CAF抗体沉淀含有P/CAF的蛋白复合物。用抗evi1、P/CAF或CtBP抗体检测沉淀蛋白。星星表示非特定波段。B、用四种表达载体（EVI1、P/CAF、CtBP2、HDAC1）转染后，用抗EVI1、抗P/CAF、抗CtBP2、抗HDAC1抗体免疫沉淀福尔马林固定的DNA片段。免疫沉淀后，纯化的DNA在GATA2启动子区通过特定的引物对扩增。各种特异性IgG作为同型对照进行免疫沉淀。C， EVI1-K564A突变体转染后GATA2转录激活的丧失。将EVI1或EVI1- k564a突变体表达载体分别与CtBP2单独或CtBP2结合P/CAF表达载体转染COS7细胞，荧光素酶检测GATA2启动子活性。GATA2转染赖氨酸突变的EVI1载体。然后，用特异性EVI1结合E， P/CAF EVI1 EVI1 GATA2 EVI1 CtBP2对照F共转染EVI1或EVI1- k564a突变体表达载体进入COS7细胞，用抗flag M2抗体纯化的EVI1结合D1/b或D2/i特异性寡核苷酸进行GMSA。用抗flag - m2抗体检测纯化的EVI1蛋白，并在GMSA反应中加入100倍过量的冷结合寡核苷酸（D1/b或D2/i）。G、采用半定量RT-PCR方法检测shP/CAF表达过程中UCSD/AML1细胞中EVI1、P/CAF和GATA2的表达水平。以带shLuc表达载体的UCSD/AML1细胞为对照。H，在shLuc和shP/CAF的UCSD/AML1细胞中，使用b-actin作为对照的每种特异性抗体，通过免疫印迹检测P/CAF水平、lys564位点EVI1乙酰化水平和EVI1水平。I，将shP/CAF或shLuc表达载体转染UCSD/AML1细胞后，对GATA2启动子区EVI1结合位点的染色质免疫沉淀分析。用抗evi1抗体或对照小鼠IgG沉淀DNA。
were transfected into COS7 cells, and protein complexes containing P/CAF were precipitated by anti-P/CAF antibodies. Precipitated proteins were detected by anti-EVI1, P/CAF or CtBP antibodies. Stars indicate nonspecific bands. B , After transfection with four kinds of expression vectors (EVI1, P/CAF, CtBP2, or HDAC1), formalin-fixed DNA fragments were immunoprecipitated with anti-EVI1, anti-P/CAF, anti-CtBP2, or anti-HDAC1 antibody. After immunoprecipitation, purified DNA was amplified by specific pairs of primers in the GATA2 promoter region. Each species-specific IgG was used for immunoprecipitation as an isotype control. C , Loss of GATA2 transcriptional activation by transfection with an EVI1-K564A mutant. EVI1 or EVI1-K564A mutant expression vectors were transfected into COS7 cells either with CtBP2 alone or CtBP2 with P/CAF expression vectors for luciferase assays of GATA2 promoter activity. GATA2 transfection the lysine-mutated EVI1 vector. After transfection with mutants GATA2 control Then, using specific EVI1 binding E , P/CAF EVI1 EVI1 GATA2 After co-transfection EVI1 CtBP2 control F, Gel-mobility shift assay EVI1 or EVI1-K564A mutant expression vectors into COS7 cells, EVI1 purified by anti-FLAG M2 antibody was used for GMSA with EVI1 binding oligonucleotides specific for D1/b or D2/i. Purified EVI1 protein was detected with anti-FLAG-M2 antibody, and a 100-fold excess of cold binding oligonucleotides (D1/b or D2/i) was added to the GMSA reaction. G , Levels of EVI1, P/CAF and GATA2 were determined in UCSD/AML1 cells during shP/CAF expression by semi-quantitative RT-PCR. UCSD/AML1 cells with an shLuc expression vector were used as a control. H, Levels of P/CAF, EVI1 acetylation at Lys 564 , and EVI1 were determined by immunoblot using each specific antibody with b-actin as a control in both UCSD/AML1 cells with shLuc and with shP/CAF. I, Chromatin-immunoprecipitation analysis of the EVI1 binding site in the GATA2 promoter region after transfection of shP/CAF or shLuc expression vectors into UCSD/AML1 cells. DNA was precipitated with an anti-EVI1 antibody or control mouse IgG.