Inhibition of SIRT1 increases EZH2 protein level and enhances the repression of EZH2 on target gene expression / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To study the regulatory rolesof SIRT1 on EZH2 expression and the further effects on EZH 2’ s repression of target gene expression.</p><p><b>METHODS</b>The stable SIRT1 RNAi and Control RNAi HeLa cells were established by infection with retroviruses expressing shSIRT1 and shLuc respectively followed by puromycin selection. EZH2 protein level was detected by Western blot in either whole cell lysate or the fractional cell extract. Reverse transcription-polymerase chain reaction was performed to detect the mRNA level of EZH2. Cycloheximide was used to treat SIRT1 RNAi and Control RNAi cells for protein stability assay. Chromatin immunoprecipitation(ChIP) assay was applied to measure enrichment of SIRT1, EZH2, and trimethylated H3K27 (H3K27me3) at SATB1 promoter in SIRT1 RNAi and Control RNAi cells.</p><p><b>RESULTS</b>Western blot results showed that EZH2 protein level increased upon SIRT1 depletion. Fractional extraction results showed unchanged cytoplasmic fraction and increased chromatin fraction of EZH2 protein in SIRT1 RNAi cells. The mRNA level of EZH2 was not affected by knockdown of SIRT1. SIRT1 recruitment was not detected at the promoter regionof EZH2 gene locus. The protein stability assay showed that the protein stability of EZH2 increases upon SIRT1 knockdown. Upon SIRT1 depletion, EZH2 and H3K27me3 recruitment at SATB1 promoter increases and the mRNA level of SATB1 decreases.</p><p><b>CONCLUSIONS</b>Depletion of SIRT1 increases the protein stability of EZH2. The regulation of EZH2 protein level by SIRT1 affects the repressive effects of EZH2 on the target gene expression.</p>

NF-E2: a novel regulator of alpha-hemoglobin stabilizing protein gene expression.

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 ß-thalassemia treatment.

Epigenetic repression of SATB1 by polycomb group protein EZH2 in epithelial cells.

OBJECTIVE: To study the regulatory mechanism of SATB1 repression in cells other than T cells or erythroid cells, which have high expression level of SATB1. METHODS: HeLa epithelial cells were treated with either histone deacetylase inhibitor (HDACi) trichostatin A (TSA) or DNA methylation inhibitor 5-Aza-C before detecting SATB1 expression. Luciferase reporter system was applied to measure effects of EZH2 on SATB1 promoter activity. Over-expression or knockdown of EZH2 and subsequent quantitative reverse transcription-polymerase chain reaction were performed to determine the effect of this Polycomb group protein on SATB1 transcription. Chromatin immunoprecipitation (ChIP) assay was applied to measure enrichment of EZH2 and trimethylated H3K27 (H3K27me3) at SATB1 promoter in HeLa cells. K562 cells and Jurkat cells, both having high-level expression of SATB1, were used in the ChIP experiment as controls. RESULTS: Both TSA and 5-Aza-C increased SATB1 expression in HeLa cells. Over-expression of EZH2 reduced promoter activity as well as the mRNA level of SATB1, while knockdown of EZH2 apparently enhanced SATB1 expression in HeLa cells but not in K562 cells and Jurkat cells. ChIP assay Results suggested that epigenetic silencing of SATB1 by EZH2 in HeLa cells was mediated by trimethylation modification of H3K27. In contrast, enrichment of EZH2 and H3K27me3 was not detected within proximal promoter region of SATB1 in either K562 or Jurkat cells. CONCLUSION: SATB1 is a bona fide EZH2 target gene in HeLa cells and the repression of SATB1 by EZH2 may be mediated by trimethylation modification on H3K27.

Regulation of acyl-coenzyme A: cholesterol acyltransferase 2 expression by saturated fatty acids.

OBJECTIVE: To verify the regulation of acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT 2), which is associated with cholesterol metabolism, by saturated fatty acids (SFAs). METHODS: Palmitic acid (PA), the most abundant saturated fatty acid in plasma, and oleic acid (OA), a widely distributed unsaturated fatty acid, were used to treat hepatic cells HepG2, HuH7, and mouse primary hepatocytes. In addition, PA at different concentrations and PA treatment at different durations were applied in HepG2 cells. In in vivo experiment, three-month male C57/BL6 mice were fed with control diet and SFA diet containing hydrogenated coconut oil rich of SFAs. The mRNA level of ACAT2 in those hepatic cells and the mouse livers was detected with real-time polymerase chain reaction (PCR). RESULTS: In the three types of hepatic cells treated with PA, that SFA induced significant increase of ACAT2 expression (Pü0.01), whereas treatment with OA showed no significant effect. That effect of PA was noticed gradually rising along with the increase of PA concentration and the extension of PA treatment duration (both Pü0.05). SFA diet feeding in mice resulted in a short-term and transient increase of ACAT2 expression in vivo, with a peak level appearing in the mice fed with SFA diet for two days (Pü0.05). CONCLUSION: SFA may regulate ACAT2 expression in human and mouse hepatic cells and in mouse livers.

Gaussia luciferase reporter assay for assessment of gene delivery systems in vivo / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To develop an alternative method for assessment of gene delivery systems in vivo.</p><p><b>METHODS</b>Mouse primary spleen lymphocytes were genetically modified in vitro by a retroviral vector harboring a Gaussia luciferase (Gluc) expression cassette. After implantation of these cells into recipient mice, the expression of Gluc was detected in whole blood or plasma collected.</p><p><b>RESULTS</b>As little as 10 muL whole blood drawn from the recipient mice could guarantee prompt reading of Gluc activity with a luminometer. And the reading was found in good correlation with the number of genetically modified spleen lymphocytes implanted to the mice.</p><p><b>CONCLUSIONS</b>Gluc may be useful as an in vivo reporter for gene therapy researches, and Gluc blood assay could provide an alternative method for assessment of gene delivery systems in vivo.</p>

Establishment of BAC mediated transgenic mice containing 97 kb beta-globin gene cluster / 中国医学科学院学报

<p><b>OBJECTIVE</b>To delete IL-11 receptor alpha chain gene from the Bacterial Artificial Chromosome (BAC) chimeric DNA by RecA protein mediated homologous recombination method and establish the transgenic mice model containing whole beta-globin gene cluster.</p><p><b>METHODS</b>Two 500 bp homologous sequences (A and B) located at the upstream and downstream of IL-11 receptor alpha chain gene respectively were cloned into the Hind III and Xba I sites of pBV vector, then the 1 kb A + B fragment was recovered from the building vector and inserted into the Sal I site of the shuttle vector pSV-RecA. After transforming the shuttle vector into the competent DH10B E. Coli containing BAC DNA, the IL-11 receptor alpha chain gene was finally deleted from the BAC DNA through chloramphenicol positive selection and fusaic acid negative selection. The new BAC clone was characterized by Pulse Field Gel Electrophoresis (PFGE). Then, we microinjected the linearized and purified BAC DNA into the mouse fertilized eggs and prepared the transgenic mice.</p><p><b>RESULTS</b>By RecA protein mediated homologous recombination method, we deleted the IL-11 receptor alpha chain gene from the BAC DNA containing the complete beta-globin Gene Cluster and established 3 respective transgenic mice lines.</p><p><b>CONCLUSION</b>Human beta-globin gene cluster in the transgenic mice mediated by new BAC expresses in a correct mode and level as compared with previous transgenic mice.</p>