Sustained expression of the Spi-1/PU.1 and Fli-1 oncoproteins blocks globin gene activation in mouse erythroleukemia cells; however, only Spi-1/PU.1 expression inhibits the inclusion of exon 16 in the mature 4.1R mRNA...Sustained expression of the Spi-1/PU.1 and Fli-1 oncoproteins blocks globin gene activation in mouse erythroleukemia cells; however, only Spi-1/PU.1 expression inhibits the inclusion of exon 16 in the mature 4.1R mRNA. This splicing event is crucial for a functional 4.1R protein and, therefore, for red blood cell membrane integrity. This report demonstrates that Spi-1/PU.1 downregulation induces the activation of TRIM10/hematopoietic RING finger 1 （HERF1）, a member of the tripartite motif （TRIM）/RBCC protein family needed for globin gene transcription. Additionally, we demonstrate that TRIM10/HERF1 is required for the regulated splicing of exon 16 during late erythroid differentia- tion. Using inducible overexpression and silencing approaches, we found that： （1） TRIM10/HERF1 knockdown inhibits hemoglobin production and exon splicing and triggers cell apoptosis in dimethylsulfoxide （DMSO）-induced cells; （2） TRIM10/HERF1 upregulation is required but is insufficient on its own to activate exon retention; （3） Fli-1 has no effect on TRIM10/HERFI expression, whereas either DMSO-induced downregulation or shRNA-knockdown of Spi-I/PU.I expression is sufficient to activate TRIM10/HERF1 expression; and （4） Spi-1/PU.1 knockdown triggers both the transcription and the splicing events independently of the chemical induction. Altogether, these data indicate that primary Spi-1/PU.1 downregulation acts on late erythroid differentiation through at least two pathways, one of which requires TRIM10/HERF1 upregulation and parallels the Spi-1/PU.1-induced Fli-1 shutoff regulatory cascade.展开更多
AIM:To investigate the function of PU.1-silenced semimature dendritic cells(DCs)and the possibility of utilizing cell immunity in rat intestinal transplantation.METHODS:DCs were isolated from the bone marrow of F344 r...AIM:To investigate the function of PU.1-silenced semimature dendritic cells(DCs)and the possibility of utilizing cell immunity in rat intestinal transplantation.METHODS:DCs were isolated from the bone marrow of F344 rats and cultured using the adherent method.The PU.1 gene was knocked down in DCs using small interfering RNAs(siRNAs)for 24 h,and the cells were then incubated with lipopolysaccharide for 48 h.The PU.1 siRNA that had the highest silencing efficiency was screened using reverse transcription-polymerase chain reaction and Western blot for further study.The tolerance capacity was analyzed and compared between PU.1-silenced DCs(siRNA PU.1 group),negative control-silenced DCs(siRNA NC group)and immature DCs(control group)both in vitro and in vivo.CONCLUSION:Blocking expression of the PU.1 gene in vitro led to a reduction in DC maturation and an increased tolerance capability.PU.1-silenced DCs expressed moderate levels of major histocompatibility complex(MHC)-Ⅱand low levels of co-stimulatory molecules,and produced more interleukin(IL)-10,but less IL-12.Compared with the negative control group,surface molecules cluster of differentiation 80(CD80),CD86 and MHC-Ⅱin the siRNA PU.1 group were 27.0%±5.6%,23.6%±4.8%and 36.8%±6.8%,respectively,and showed a significantly lower trend(P<0.05).In vivo treatment of recipients with PU.1-silenced DCs injected before intestinal transplantation(siRNA PU.1group),significantly prolonged allograft survival and resulted in better tissue histopathology compared with the siRNA NC group and control group.Mean survival time after transplantation was 14.3±3.3 d in the siRNA PU.1 group(P<0.05).CONCLUSION:PU.1-silenced semi-mature DCs induced partial immune tolerance both in vitro and in vivo,which could be used as a new strategy to promote transplantation tolerance.展开更多
文摘Sustained expression of the Spi-1/PU.1 and Fli-1 oncoproteins blocks globin gene activation in mouse erythroleukemia cells; however, only Spi-1/PU.1 expression inhibits the inclusion of exon 16 in the mature 4.1R mRNA. This splicing event is crucial for a functional 4.1R protein and, therefore, for red blood cell membrane integrity. This report demonstrates that Spi-1/PU.1 downregulation induces the activation of TRIM10/hematopoietic RING finger 1 （HERF1）, a member of the tripartite motif （TRIM）/RBCC protein family needed for globin gene transcription. Additionally, we demonstrate that TRIM10/HERF1 is required for the regulated splicing of exon 16 during late erythroid differentia- tion. Using inducible overexpression and silencing approaches, we found that： （1） TRIM10/HERF1 knockdown inhibits hemoglobin production and exon splicing and triggers cell apoptosis in dimethylsulfoxide （DMSO）-induced cells; （2） TRIM10/HERF1 upregulation is required but is insufficient on its own to activate exon retention; （3） Fli-1 has no effect on TRIM10/HERFI expression, whereas either DMSO-induced downregulation or shRNA-knockdown of Spi-I/PU.I expression is sufficient to activate TRIM10/HERF1 expression; and （4） Spi-1/PU.1 knockdown triggers both the transcription and the splicing events independently of the chemical induction. Altogether, these data indicate that primary Spi-1/PU.1 downregulation acts on late erythroid differentiation through at least two pathways, one of which requires TRIM10/HERF1 upregulation and parallels the Spi-1/PU.1-induced Fli-1 shutoff regulatory cascade.
基金Supported by a grant from the Natural Science Foundation of Jiangsu Province,China,No.BK2008237
文摘AIM:To investigate the function of PU.1-silenced semimature dendritic cells(DCs)and the possibility of utilizing cell immunity in rat intestinal transplantation.METHODS:DCs were isolated from the bone marrow of F344 rats and cultured using the adherent method.The PU.1 gene was knocked down in DCs using small interfering RNAs(siRNAs)for 24 h,and the cells were then incubated with lipopolysaccharide for 48 h.The PU.1 siRNA that had the highest silencing efficiency was screened using reverse transcription-polymerase chain reaction and Western blot for further study.The tolerance capacity was analyzed and compared between PU.1-silenced DCs(siRNA PU.1 group),negative control-silenced DCs(siRNA NC group)and immature DCs(control group)both in vitro and in vivo.CONCLUSION:Blocking expression of the PU.1 gene in vitro led to a reduction in DC maturation and an increased tolerance capability.PU.1-silenced DCs expressed moderate levels of major histocompatibility complex(MHC)-Ⅱand low levels of co-stimulatory molecules,and produced more interleukin(IL)-10,but less IL-12.Compared with the negative control group,surface molecules cluster of differentiation 80(CD80),CD86 and MHC-Ⅱin the siRNA PU.1 group were 27.0%±5.6%,23.6%±4.8%and 36.8%±6.8%,respectively,and showed a significantly lower trend(P<0.05).In vivo treatment of recipients with PU.1-silenced DCs injected before intestinal transplantation(siRNA PU.1group),significantly prolonged allograft survival and resulted in better tissue histopathology compared with the siRNA NC group and control group.Mean survival time after transplantation was 14.3±3.3 d in the siRNA PU.1 group(P<0.05).CONCLUSION:PU.1-silenced semi-mature DCs induced partial immune tolerance both in vitro and in vivo,which could be used as a new strategy to promote transplantation tolerance.
