Objective To investigate the role of lysine-specific demethylase 1 （LSD1） in the process of THP-1 monocyte-to-macrophage differentiation. Methods Quantitative reverse transcription-polymerase chain reaction （qRT-PCR） and Western blotting were performed to analyze the expression of LSD1 and interleukin-6 （IL-6） in THP-1 monocytes and THP-l-derived macrophages. Chromatin immunoprecipitation （ChiP） assay was applied to detect the occupancy of LSD1 and H3K4 methylation at IL-6 promoter during THP-1 monocyte-to-macrophage differentiation. IL-6 mRNA level and H3K4 methylation at IL-6 promoter were analyzed using qRT-PCR and ChiP assay in LSD 1 -knockdown THP- 1 cells treated with 12-O-tetradecanoylphorbol- 13-acetate （TPA） for 0 4, 8, 12, and 24 hours. Fluorescence activated flow cytometry was performed to reveal the percentage of macrophages differentiated from THP- 1 monocytes. Results The expression of LSD1 reduced during THP-1 monocyte-to-macrophage differentiation （P〈0.01）. LSD1 occupancy decreased and H3K4 methylation increased at IL-6 promoter during the differentiation. With knockdown of LSD1, H3K4 methylation at IL-6 promoter was found increased after TPA treatment at different times points （all P〈0.05, except 24 hours）. The percentage of macrophages increased significantly in theTHP-I cells with LSD1 knockdown （P〈0.05）. Conclusions LSD1 is repressed during the monocyte-to-macrophage differentiation of THP-1 cells. Suppression of LSD 1-mediated H3K4 demethylation may be required for THP-1 monocyte-to-macrophage differentiation.
Rui-feng YangGuo-wei ZhaoShu-ting LiangHou-zao ChenDe-pei Liu
DNA double-strand breaks are repaired through either non-homologous end joining(NHEJ) or homologous recombination repair(HRR) pathway.The well-characterized regulatory mechanisms of double-strand break repair(DSBR) are mainly found at the level of complicated repair protein interactions and modifications.Regulation of DSBR at the transcriptional level was also reported.In this study,we found that DSBR can be regulated by miR-34a at the post-transcriptional level.Specifically,miR-34a,which can be activated by DNA damages,represses DSBR activities by impairing both NHEJ and HRR pathways in cultured cells.The repression is mainly through targeting the critical DSBR promoting factor SIRT1,as ectopically expressed SIRT1 without 3'-UTR can rescue the inhibitory roles of miR-34a on DSBR.Further studies demonstrate that SIRT1 conversely represses miR-34a expression.Taken together,our data show that miR-34a is a new repressor of DSBR and the mutual inhibition between miR-34a and SIRT1 may contribute to regulation of DNA damage repair.
Studies on the chaperone protein α-hemoglobin stabilizing protein （AHSP） reveal that abundant AHSP in erythroid cells en-hance the cells＇ tolerance to oxidative stress imposed by excess a-hemoglobin in pathological conditions. However, the poten-tial intracellular modulation of AHSP expression itself in response to oxidative stress is still unknown. The present study ex-amined the effect and molecular mechanism of STAT3, an oxidative regulator, on the expression of AHSP. AHSP expression increased in K562 cells upon cytokine IL-6-induced STAT3 activation and decreased in STAT3 knock-down K562 cells. Reg-ulation of AHSP in oxidative circumstance was then examined in α-globin-overloaded K562 cells, and real-time PCR showed strengthened expression of both AHSP and STAT3. ChIP analysis showed binding of STAT3 to AHSP promoter and binding was significantly augmented with IL6 stimulation and upon α-globin overexpression. Dual luciferase reporter assays of the wildtype and mutated SB3 element, an IL-6RE site, in the AHSP promoter in K562 cells highlighted the direct regulatory ef-fect of STAT3 on AHSP gene. Finally, direct binding of STAT3 to SB3 site of AHSP promoter was confirmed with EMSA as-says. Our work reveals an adaptive AHSP regulation mediated by the redox-sensitive STAT3 signaling pathway, and provides clues to the therapeutic strategy for AHSP enhancement.
CAO CongZHAO GuoWeiYU WeiXIE XueMinWANG WenTianYANG RuiFengLV XiangLIU DePei
Activation of B-cells is initiated by the ligation of B-cell receptors by its cognate antigen, inducing a series of signal cascades. Understanding the molecular mechanisms of these important events is a crucial goal for immunologists. Chimeric B cell re- ceptors provide a powerful tool for analysis of B-cell signal function. However, this method can only be used in tool cells, but cannot be used for in vivo study. Here, we constructed a retroviral vector to encode both heavy chains and light chains of a membrane immunoglobulin, and expressed them in primary B-cells using retroviral gene transfer. Our results demonstrate that the membrane immunoglobulin expressed by retroviral vectors transfer can initiate B-cell receptor-mediated signaling, result- ing in the phosphorylation of Syk and Erkl/2 proteins. The results showed that B-cells expressing membrane immunoglobulin can make proliferative responses to cognate antigen both in vitro and in vivo. Therefore, we provide a methodology for rapidly analyzing the downstream signals of B-cell receptors both in vitro and in vivo, which could expedite the identification of proteins involved in B-cell function.
