LHP1 mediates recruitment of the PRC2 histone methyltransferase complex to chromatin and thereby facilitates maintenance of H3K27me3 on FLC, a key flowering repressor gene. Here, we report that the PWWP domain protei...LHP1 mediates recruitment of the PRC2 histone methyltransferase complex to chromatin and thereby facilitates maintenance of H3K27me3 on FLC, a key flowering repressor gene. Here, we report that the PWWP domain proteins （PDPs） interact with FVE and MSI5 to suppress FLC expression and thereby promote flowering. We demonstrated that FVE, MSI5, and PDP3 were co-purified with LHP1. The H3K27me3 level on FLC was decreased in the pdp mutants as welt as in the fve/ msi5 double mutant. This study suggests that PDPs function together with FVE and MSI5 to regulate the function of the PRC2 complex on FLC.展开更多
The effects of epigenetic modification on the differentiation of islet cells and the expression of associated genes（Pdx-1,Pax4,MafA,and Nkx6.1,etc） were investigated.The promoter methylation status of islet differen...The effects of epigenetic modification on the differentiation of islet cells and the expression of associated genes（Pdx-1,Pax4,MafA,and Nkx6.1,etc） were investigated.The promoter methylation status of islet differentiation-associated genes（Pdx-1,Pax4,MafA and Nkx6.1）,Oct4 and MLH1 genes of mouse embryonic stem cells,NIH3T3 cells and NIT-1 cells were profiled by methylated DNA immunoprecipitation,real-time quantitative PCR（MeDIP-qPCR） techniques.The histone modification status of these genes promoter region in different cell types was also measured by using chromatin immunoprecipitation real-time quantitative PCR methods.The expression of these genes in these cells was detected by using real-time quantitative PCR.The relationship between the epigenetic modification（DNA methylation,H3 acetylation,H3K4m3 and H3K9m3） of these genes and their expression was analyzed.The results showed that：（1） the transcription-initiation-sites of Pdx-1,MafA and Nkx6.1 were highly methylated in NIH3T3 cells; （2） NIH3T3 cells showed a significantly higher level of DNA methylation modification in the transcription-initiation-site of Pdx-1,Pax4,MafA and Nkx6.1 genes than that in mES cells and NIT-1 cells（P〈0.05）; （3） NIT-1 cells had a significantly higher level of H3K4m3 modification in the transcription-initiation-site of Pdx-1,Pax4,MafA and Nkx6.1 genes than that in mES cells and NIH3T3 cells（P〈0.05）,with significantly increased level of gene expression; （4） NIH3T3 cell had a significantly higher level of H3K9m3 modification in the transcription-initiation-site of Pdx-1,Pax4,MafA and Nkx6.1 genes than that in mES cells and with NIT-1 cell（P〈0.05）,with no detectable mRNA expression of these genes.It was concluded that histone modification（H3K4m3 and H3K9m3） and DNA methylation might have an intimate communication between each other in the differentiation process from embryonic stem cells into islet cells.展开更多
Objective To investigate whether α-hemoglobin stabilizing protein (AHSP), the α-globin-specific molecular chaperone, is regulated by erythroid transcription factor NF-E2. Methods We established the stable cell line ...Objective To investigate whether α-hemoglobin stabilizing protein (AHSP), the α-globin-specific molecular chaperone, is regulated by erythroid transcription factor NF-E2. Methods We established the stable cell line with NF-E2p45 (the larger subunit of NF-E2) short hairpin RNA to silence its expression. Western blot, real-time polymerase chain reaction, and chromatin immunoprecipitation (ChIP) analysis were performed to detect the expression of AHSP, the histone modifications at AHSP gene locus, and the binding of GATA-1 at the AHSP promoter with NF-E2p45 deficiency. ChIP was also carried out in dimethyl sulfoxide (DMSO)-induced DS19 cells and estrogen-induced G1E-ER4 cells to examine NF-E2 binding to the AHSP gene locus and its changes during cell erythroid differentiation. Finally, luciferase assay was applied in HeLa cells transfected with AHSP promoter fragments to examine AHSP promoter activity in the presence of exogenous NF-E2p45. Results We found that AHSP expression was highly dependent on NF-E2p45. NF-E2 bound to the regions across AHSP gene locus in vivo, and the transcription of AHSP was transactivated by exogenous NF-E2p45. In addition, we observed the decrease of H3K4 trimethylation and GATA-1 occupancy at the AHSP gene locus in NF-E2p45-deficient cells. Restoration of GATA-1 in G1E-ER4 cells in turn led to increased DNA binding of NF-E2p45. Conclusion NF-E2 may play an important role in AHSP gene regulation, providing new insights into the molecular mechanisms underlying the erythroid-specific expression of AHSP as well as new possibilities for β-thalassemia treatment.展开更多
The mammalian protein kinase C-interacting cousin of thioredoxin(PICOT;also termed glutaredoxin 3)is a multi-domain monothiol glutaredoxin that is involved in a wide variety of signaling pathways and biological proces...The mammalian protein kinase C-interacting cousin of thioredoxin(PICOT;also termed glutaredoxin 3)is a multi-domain monothiol glutaredoxin that is involved in a wide variety of signaling pathways and biological processes.PICOT is required for normal and transformed cell growth and is critical for embryonic development.Recent studies in T lymphocytes demonstrated that PICOT can translocate to the nucleus and interact with embryonic ectoderm development,a polycomb group protein and a core component of the polycomb repressive complex 2,which contributes to the maintenance of transcriptional repression and chromatin remodeling.Furthermore,PICOT was found to interact with chromatin-bound embryonic ectoderm development and alter the extent of histone 3 lysine 27 trimethylation at the promoter region of selected polycomb repressive complex 2 target genes.PICOT knockdown in Jurkat T cells led to increased histone 3 lysine 27 trimethylation at the promoter region of CCND2,a cell cycle-regulating gene which encodes the cyclin D2 protein.As a result,the expression levels of CCND2 mRNA and protein levels were reduced,concomitantly with inhibition of the cell growth rate.Analysis of multiple data sets from the Cancer Genome Atlas revealed that a high expression of PICOT correlated with a low expression of CCND2 in a large number of human cancers.In addition,this parameter correlated with poor patient survival,suggesting that the ratio between PICOT/CCND2 mRNA levels might serve as a predictor of patient survival in selected types of human cancer.展开更多
目的通过比较不同细胞类型之间胰腺十二指肠同源盒1（Pdx-1）、配对盒基因4（Pax4）、MafA（mast cell function associated antigen）和Nkx6．1等胰岛组织特异性基因其转录起始区的H3K4m3和H3K9m3修饰的差异，探讨H3K4nd和H3K9m3修饰...目的通过比较不同细胞类型之间胰腺十二指肠同源盒1（Pdx-1）、配对盒基因4（Pax4）、MafA（mast cell function associated antigen）和Nkx6．1等胰岛组织特异性基因其转录起始区的H3K4m3和H3K9m3修饰的差异，探讨H3K4nd和H3K9m3修饰对胰岛组织特异性基因表达的作用。方法采用染色质免疫共沉淀．实时定量聚合酶链反应（PCR）法检测小鼠胚胎干细胞（mES，1×10^7）、小鼠成纤维细胞株NIH3T3细胞（1×10^7）和小鼠B细胞株NIT-1细胞（1×10^7）三者中的胰岛组织特异性基因、Oct4基因和MLH1基因转录起始区H3K4rrd和H3K9m3修饰的状况。同时采用实时定量逆转录（RT）-PCR检测上述3种细胞各基因mRNA表达水平。分析H3K4m3和H3K9m3修饰改变与基因表达之间的关系。结果NIT-1细胞中Pdx-1、Pax4、MafA、Nkx6．1等胰岛组织特异性基因转录起始区的H3K4m的修饰水平分别为：（4．84±0．05）％、（9．91±1．33）％、（10．64±0．87）％、（0．23±0．03）％，与mES细胞比较明显增高（P〈0．05），基因表达；NIH3T3细胞中Pdx-1、Pax4、MafA、Nkx6．1等胰岛组织特异性基因转录起始区的H3Kgm3的修饰水平分别为：（0．64±0．21）％、（7．04±1．29）％、（0．39±0．10）％、（2．35±0．81）％，与mES细胞比较明显增高（P〈0．05），基因不表达。结论mK4n13与H3K9m3修饰能相互协调，共同调控胰岛组织特异性基因的表达。展开更多
基金supported by the National Key Research and Development Program of China (2016YFA0500801)the 973 Program (2011CB812600) from the Chinese Ministry of Science and Technology (to X.J.H.)the National Natural Science Foundation of China (31370317 and 31571584 to B.W.)
文摘LHP1 mediates recruitment of the PRC2 histone methyltransferase complex to chromatin and thereby facilitates maintenance of H3K27me3 on FLC, a key flowering repressor gene. Here, we report that the PWWP domain proteins （PDPs） interact with FVE and MSI5 to suppress FLC expression and thereby promote flowering. We demonstrated that FVE, MSI5, and PDP3 were co-purified with LHP1. The H3K27me3 level on FLC was decreased in the pdp mutants as welt as in the fve/ msi5 double mutant. This study suggests that PDPs function together with FVE and MSI5 to regulate the function of the PRC2 complex on FLC.
文摘The effects of epigenetic modification on the differentiation of islet cells and the expression of associated genes（Pdx-1,Pax4,MafA,and Nkx6.1,etc） were investigated.The promoter methylation status of islet differentiation-associated genes（Pdx-1,Pax4,MafA and Nkx6.1）,Oct4 and MLH1 genes of mouse embryonic stem cells,NIH3T3 cells and NIT-1 cells were profiled by methylated DNA immunoprecipitation,real-time quantitative PCR（MeDIP-qPCR） techniques.The histone modification status of these genes promoter region in different cell types was also measured by using chromatin immunoprecipitation real-time quantitative PCR methods.The expression of these genes in these cells was detected by using real-time quantitative PCR.The relationship between the epigenetic modification（DNA methylation,H3 acetylation,H3K4m3 and H3K9m3） of these genes and their expression was analyzed.The results showed that：（1） the transcription-initiation-sites of Pdx-1,MafA and Nkx6.1 were highly methylated in NIH3T3 cells; （2） NIH3T3 cells showed a significantly higher level of DNA methylation modification in the transcription-initiation-site of Pdx-1,Pax4,MafA and Nkx6.1 genes than that in mES cells and NIT-1 cells（P〈0.05）; （3） NIT-1 cells had a significantly higher level of H3K4m3 modification in the transcription-initiation-site of Pdx-1,Pax4,MafA and Nkx6.1 genes than that in mES cells and NIH3T3 cells（P〈0.05）,with significantly increased level of gene expression; （4） NIH3T3 cell had a significantly higher level of H3K9m3 modification in the transcription-initiation-site of Pdx-1,Pax4,MafA and Nkx6.1 genes than that in mES cells and with NIT-1 cell（P〈0.05）,with no detectable mRNA expression of these genes.It was concluded that histone modification（H3K4m3 and H3K9m3） and DNA methylation might have an intimate communication between each other in the differentiation process from embryonic stem cells into islet cells.
基金Supported by National Natural Science Foundation of China (30130026, U0632005, 30721063)National Basic Research Program of China (973 Program) (2011CB964803)+1 种基金National Laboratory of Medical Molecular Biology grant (2060204)Beijing municipal government grant (YB20081002301)
文摘Objective To investigate whether α-hemoglobin stabilizing protein (AHSP), the α-globin-specific molecular chaperone, is regulated by erythroid transcription factor NF-E2. Methods We established the stable cell line with NF-E2p45 (the larger subunit of NF-E2) short hairpin RNA to silence its expression. Western blot, real-time polymerase chain reaction, and chromatin immunoprecipitation (ChIP) analysis were performed to detect the expression of AHSP, the histone modifications at AHSP gene locus, and the binding of GATA-1 at the AHSP promoter with NF-E2p45 deficiency. ChIP was also carried out in dimethyl sulfoxide (DMSO)-induced DS19 cells and estrogen-induced G1E-ER4 cells to examine NF-E2 binding to the AHSP gene locus and its changes during cell erythroid differentiation. Finally, luciferase assay was applied in HeLa cells transfected with AHSP promoter fragments to examine AHSP promoter activity in the presence of exogenous NF-E2p45. Results We found that AHSP expression was highly dependent on NF-E2p45. NF-E2 bound to the regions across AHSP gene locus in vivo, and the transcription of AHSP was transactivated by exogenous NF-E2p45. In addition, we observed the decrease of H3K4 trimethylation and GATA-1 occupancy at the AHSP gene locus in NF-E2p45-deficient cells. Restoration of GATA-1 in G1E-ER4 cells in turn led to increased DNA binding of NF-E2p45. Conclusion NF-E2 may play an important role in AHSP gene regulation, providing new insights into the molecular mechanisms underlying the erythroid-specific expression of AHSP as well as new possibilities for β-thalassemia treatment.
基金Supported by the USA-Israel Binational Science Foundation,No.2013034the Israel Science Foundation administered by the Israel Academy of Science,No.1235/17+1 种基金the Jacki and Bruce Barron Cancer Research Scholars’ProgramCity of Hope and the Israel Cancer Research Fund,No.87735611.
文摘The mammalian protein kinase C-interacting cousin of thioredoxin(PICOT;also termed glutaredoxin 3)is a multi-domain monothiol glutaredoxin that is involved in a wide variety of signaling pathways and biological processes.PICOT is required for normal and transformed cell growth and is critical for embryonic development.Recent studies in T lymphocytes demonstrated that PICOT can translocate to the nucleus and interact with embryonic ectoderm development,a polycomb group protein and a core component of the polycomb repressive complex 2,which contributes to the maintenance of transcriptional repression and chromatin remodeling.Furthermore,PICOT was found to interact with chromatin-bound embryonic ectoderm development and alter the extent of histone 3 lysine 27 trimethylation at the promoter region of selected polycomb repressive complex 2 target genes.PICOT knockdown in Jurkat T cells led to increased histone 3 lysine 27 trimethylation at the promoter region of CCND2,a cell cycle-regulating gene which encodes the cyclin D2 protein.As a result,the expression levels of CCND2 mRNA and protein levels were reduced,concomitantly with inhibition of the cell growth rate.Analysis of multiple data sets from the Cancer Genome Atlas revealed that a high expression of PICOT correlated with a low expression of CCND2 in a large number of human cancers.In addition,this parameter correlated with poor patient survival,suggesting that the ratio between PICOT/CCND2 mRNA levels might serve as a predictor of patient survival in selected types of human cancer.
文摘目的通过比较不同细胞类型之间胰腺十二指肠同源盒1（Pdx-1）、配对盒基因4（Pax4）、MafA（mast cell function associated antigen）和Nkx6．1等胰岛组织特异性基因其转录起始区的H3K4m3和H3K9m3修饰的差异，探讨H3K4nd和H3K9m3修饰对胰岛组织特异性基因表达的作用。方法采用染色质免疫共沉淀．实时定量聚合酶链反应（PCR）法检测小鼠胚胎干细胞（mES，1×10^7）、小鼠成纤维细胞株NIH3T3细胞（1×10^7）和小鼠B细胞株NIT-1细胞（1×10^7）三者中的胰岛组织特异性基因、Oct4基因和MLH1基因转录起始区H3K4rrd和H3K9m3修饰的状况。同时采用实时定量逆转录（RT）-PCR检测上述3种细胞各基因mRNA表达水平。分析H3K4m3和H3K9m3修饰改变与基因表达之间的关系。结果NIT-1细胞中Pdx-1、Pax4、MafA、Nkx6．1等胰岛组织特异性基因转录起始区的H3K4m的修饰水平分别为：（4．84±0．05）％、（9．91±1．33）％、（10．64±0．87）％、（0．23±0．03）％，与mES细胞比较明显增高（P〈0．05），基因表达；NIH3T3细胞中Pdx-1、Pax4、MafA、Nkx6．1等胰岛组织特异性基因转录起始区的H3Kgm3的修饰水平分别为：（0．64±0．21）％、（7．04±1．29）％、（0．39±0．10）％、（2．35±0．81）％，与mES细胞比较明显增高（P〈0．05），基因不表达。结论mK4n13与H3K9m3修饰能相互协调，共同调控胰岛组织特异性基因的表达。
