The role of the cell cycle in differentiation of the cellular slime mould Dictyostelium discoideum

Summary During development and differentiation of the cellular slime mould Dictyostelium discoideum there appears to be a relationship between the cell cycle and cell fate: amoebae halted in G2 phase during early development differentiate into spores whereas stalk cells are formed from amoebae halted in GI phase. It is proposed that this is because a major effect of the cell cycle is to generate heterogeneity in the cell surface properties of the developing amoebae.     

Rapid patterning in 2-D cultures of Dictyostelium cells and its relationship to zonal differentiation

Rapid patterning has been observed in confined 2-D cultures of Dictyostelium discoideum Ax-2 cells as an outer dark zone and a inner light zone. The width of outer zone was usually approximately100 microm, irrespective of the size of cell masses under atmospheric conditions. The width of the outer zone, however, changed depending on external O2 concentrations and reached up to 250 microm at 100% O2. A clear regional difference in tetramethyl rhodamine methyl ester (TMRM) staining was noticed between the outer zone and the inner zone: the inner zone was more strongly stained with TMRM than the outer zone, which faced the air. Using inhibitors of oxidative phosphorylation (dinitrophenol (DNP) or NaN3) and a specific inhibitor of CN-resistant respiration (benzohydroxamic acid (BHAM)), it has been demonstrated that the outer zone is basically formed by the O2 threshold for oxidative phosphorylation, while the inner cells mainly perform cyanide-resistant respiration. When cells around the early mound stage (just before prestalk and prespore differentiation) were cultured as 2-D cell masses, ecmA-expressing cells (pstA cells), ecmB-expressing cells (pstB cells) and D19-expressing cells (prespore; psp cells), arose in a position-dependent manner in the outer zone. In the inner zone, cell motility seemed to be markedly impaired and neither prestalk nor prespore differentiation occurred. In addition, once-differentiated prespore cells were found to dedifferentiate rapidly in the inner zone. The reason for dedifferentiation as well as for failure of cells to differentiate in the inner zone is discussed with reference to O2 radicals.     

盘基网柄菌细胞分化和凋亡中酸性磷酸酶的定位

利用电镜酶细胞化学方法,观察盘基网柄菌细胞分化和凋亡过程中酸性磷酸酶的变化。在细胞丘阶段,酶反应颗粒出现在线粒体内自噬空泡内,随着内自噬空泡的逐渐增大,线粒体内的酶反应颗粒逐渐增多,线粒体内嵴结构不断破坏,直至遍布整个空泡化的线粒体内;当细胞发育至前孢子细胞时,由于嵴结构被完全破坏,酶反应颗粒主要集中在前孢子细胞空泡的单层膜上,空泡化的线粒体内酶反应颗粒逐渐消失。在凋亡的柄细胞中,自噬泡内酶反应强烈,凋亡中期的前柄细胞的细胞核中出现酶反应颗粒,均匀分布在细胞核中,直至细胞核与自噬泡融合。在孢子细胞外被与质膜间也观察到非溶酶体酸性磷酸酶。所得结果证实:线粒体内自噬小泡具有消化功能;自噬泡内酶活性与细胞器消亡有关;细胞核中的酸性磷酸酶可能作为一种非溶酶体酸性磷酸酶参与细胞核中核蛋白的脱磷酸化过程,与发育相关基因表达有关     

Specific expression of a gene encoding a novel calcium-binding protein, CAF-1, during transition of Dictyostelium cells from growth to differentiation

The gene expressions involved in the transition from cell proliferation to differentiation were analyzed, using synchronized Dictyostelium discoideum Ax-2 cells and the differential plaque hybridization method. As one of the genes (cDNA) specifically expressed when Ax-2 cells were starved just before the putative shift (PS)-point (putative shift point; a switchover point from growth to differentiation in the cell cycle), calfumirin-1 ( CAF-1 ) was cloned, which encoded a novel calcium-binding protein with E-F hand. Although CAF-1 mRNA was slightly expressed in vegetatively growing cells, the expression was markedly increased in response to starvation of cells just before the PS-point. Northern analysis using non-synchronized Ax-2 cells showed that the CAF-1 mRNA is predominantly expressed within a few hours of starvation. Such a starvation-induced early expression of the CAF-1 mRNA raised a possibility that CAF-1 might be one of Ca²⁺-binding proteins involved in the phase-shift of cells from growth to differentiation.     

Induction of dystrophin Dp71 expression during neuronal differentiation: opposite roles of Sp1 and AP2α in Dp71 promoter activity

In this study, we delineated the molecular mechanisms that modulate Dp71 expression during neuronal differentiation, using the N1E‐115 cell line. We demonstrated that Dp71 expression is up‐regulated in response to cAMP‐mediated neuronal differentiation of these cells, and that this induction is controlled at promoter level. Functional deletion analysis of the Dp71 promoter revealed that a 5′‐flanking 159‐bp DNA fragment that contains Sp1 and AP2 binding sites is necessary and sufficient for basal expression of this TATA‐less promoter, as well as for its induction during neuronal differentiation. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed that Sp1 and AP2α bind to their respective DNA elements within the Dp71 basal promoter. Overall, mutagenesis assays on the Sp1 and AP2 binding sites, over‐expression of Sp1 and AP2α, as well as knock‐down experiments on Sp1 and AP2α gene expression established that Dp71 basal expression is controlled by the combined action of Sp1 and AP2α, which act as activator and repressor, respectively. Furthermore, we demonstrated that induction of Dp71 expression in differentiated cells is the result of the maintenance of positive regulation exerted by Sp1, as well as of the loss of AP2α binding, which ultimately releases the promoter from repression.     

Involvements of a novel protein, DIA2, in cAMP signaling and spore differentiation during Dictyostelium development

Abstract The novel gene dia2 (differentiation-associated gene 2) was originally isolated as a gene expressed specifically in response to initial differentiation of Dictyostelium discoideum Ax-2 cells. Using dia2^AS cells in which the dia2 expression was inactivated by the antisense RNA method, DIA2 protein was found to be required for cAMP signaling during cell aggregation. During late development, the DIA2 protein changed its location from the endoplasmic reticulum (ER) to prespore-specific vacuoles (PSVs) that are specifically present in prespore cells of the slug. In differentiating prestalk cells, however, DIA2 was found to be nearly lost from the cells. Importantly, exocytosis of PSVs from prespore cells and the subsequent spore differentiation were almost completely impaired in dia2^AS cells. In addition, spore induction by externally applied 8-bromo cAMP was significantly suppressed in dia2^AS cells. Taken together, these results strongly suggested that DIA2 might be closely involved in cAMP signaling and spore differentiation as well as in the initiation of differentiation during Dictyostelium development.     

A gene encoding a novel anti-adhesive protein is expressed in growing cells and restricted to anterior-like cells during development of Dictyostelium

The Dictyostelium gene ampA, initially identified by the D11 cDNA, encodes a novel anti-adhesive-like protein. The ampA gene product inhibits premature cell agglutination during growth and modulates cell-cell and cell-substrate adhesion during development. Analysis of the promoter indicates that cap site-proximal sequence directs ampA expression during both growth and early development. Expression following tip formation is controlled by more distal sequence, which contains TTGA repeats known to regulate prestalk cell gene expression in other promoters. Comparison of reporter gene expression and endogenous mRNA accumulation indicates that during growth the ampA gene is expressed in an increasing number of cells as a function of density. The number of cells expressing the ampA gene drops as development initiates, but the cells that continue to express the gene do so at high levels. These cells are initially scattered throughout the entire aggregate. By the tip formation stage, however, the majority of ampA-expressing cells are localized to the mound periphery, with only a few cells remaining scattered in the upper portion of the mound. In the final culminant, ampA is expressed only in the upper cup, lower cup, and basal disc. Although reporter expression is observed in cells that migrate anteriorly to a banded region just posterior to the tip, expression is rarely observed in the extreme tip. AmpA protein however, is localized to the tip as well as to ALCs during late development. The results presented here suggest that ampA gene expression is shut off in ALCs that continue along the prestalk differentiation pathway before they are added to the primordial stalk.     

用于细胞水平家蚕转录调控元件研究的简单基础启动子的构建

【目的】本研究致力于构建一种能够在家蚕Bombyx mori细胞水平稳定表达的简单基础启动子,从而更准确地反映单一转录调控元件对基因启动子活性的影响,为研究家蚕乃至其他昆虫的基因转录调控奠定基础。【方法】本研究在本课题组已报道的能在家蚕细胞中稳定表达且基本不含上游转录调控元件的BmVgP78M启动子的基础上,通过PCR技术在其上游添加一定长度的间隔序列和能够应答20-羟基蜕皮激素(20E)且增强启动子活性的BrC-Z2转录因子结合基序(BrC-Z2 element, BrC-Z2E);通过基因克隆技术构建细胞转染载体;通过细胞转染技术和双荧光素酶报告基因系统检测启动子活性的变化。【结果】通过在BmVgP78M启动子上游添加28 bp间隔序列,成功构建了一个简单基础启动子,命名为VgP78ML,并证明其为可用于研究目标转录调控元件的简单基础启动子。经实验验证表明,该简单基础启动子不仅可以在家蚕细胞中稳定表达,且其本身活性不受20E及转录因子BrC-Z2的影响;当该启动子上游连接BrC-Z2E时,可以显著地应答20E及BrC-Z2转录因子,从而调控报告基因的表达。【结论】VgP78ML能够作为简单基础启动子应用于细胞水平对家蚕基因转录调控进行研究。同时,其构建方法也为其他物种构建研究转录调控的简单基础启动子提供了参考。     

Insertion of core CpG island element into human CMV promoter for enhancing recombinant protein expression stability in CHO cells

The human cytomegalovirus promoter (hCMV) is susceptible to gene silencing in CHO cells, most likely due to epigenetic events, such as DNA methylation and histone modifications. The core CpG island element (IE) from the hamster adenine phosphoribosyltransferase gene has been shown to prevent DNA methylation. A set of modified hCMV promoters was developed by inserting one or two copies of IE in either forward or reverse orientations either upstream of the hCMV enhancer, between the enhancer and core promoter (CP), or downstream of the CP. The modified hCMV with one copy of IE inserted between the enhancer and core promoter in reverse orientation (MR1) was most effective at enhancing expression stability without compromising expression level when compared with the wild‐type (WT) hCMV. A third of 18 EGFP expressing clones generated using MR1 retained 70% of their starting expression level after 8 weeks of culture in the absence of selection pressure, while none of 18 WT hCMV generated clones had expression above 50%. MR1 also improved antibody expression stability of methotrexate (MTX) amplified CHO cell lines. Stably transfected pools generated using MR1 maintained 62% of their original monoclonal antibody titer after 8 weeks of culture in the absence of MTX, compared to only 37% for WT hCMV pools. Low levels of CpG methylation within both WT hCMV and MR1 were observed in all the analyzed cell lines and the methylation levels did not correlate to the expression stability, suggesting IE enhances expression stability by other mechanisms other than preventing methylation. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:523–534, 2014     

脂肪细胞分化相关转录因子的结构和功能

关于脂肪细胞分化调控的研究主要集中在转录因子的作用上。目前了解得比较清楚的分化转录因子有多种,其中CAAT增强子结合蛋白家族（C/EBPs）中的C／EBPα和过氧化物酶体增殖物激活受体家族（PPARs）中的PPARy是转录调控中起主要作用的两种因子。两者有各自的结构特点和功能,但在脂肪细胞中它们之间相互协同促进细胞的分化成熟。本文主要就C／EBPs和PPARs家族中主要成员的结构和功能及相互作用进行综述。     

Promoter-specific function of the TATA element in undifferentiated P19 cells

P19 embryonal carcinoma cells differentiate into neuronal cells when treated with retinoic acid (RA). To explore the importance of core promoter structures in the regulation of gene expression during neuronal differentiation, the activities of three classes of modified or unmodified model promoters (Spec2a, OtxE, and Ars) were compared in P19 cells before and after RA treatment. The Spec2a promoter was activated in undifferentiated cells specifically when the E-box was located at a proximal position, whereas the OtxE promoter was activated when the E-box was in a distal position. The Ars promoter was only slightly activated by this element. In addition, the TATA element reduced the level of activation provided by the E-box, but only when it was located in the Spec2a core promoter. These results indicate that the core promoter structure may govern, at least in part, the stage-specific expression of endogenous genes involved in the neuronal differentiation of P19 cells.     

Evidence that phosphatidylcholine-specific phospholipase C is a key molecule mediating insulin-induced enhancement of gene expression from human cytomegalovirus promoter in CHO cells

The signal transduction from insulin to its receptors and Ras has been extensively studied, while little has been reported beyond these steps. We found that the expression of human interleukin 6 gene under the control of immediate early gene promoter of human cytomegalovirus was enhanced by insulin sitmulation in Chinese hamster ovary cells. The induction effect of insulin was not significantly affected by inhibitors or activators of conventional protein kinase C, cAMP dependent protein kinase and phosphoinositide -3 kinase, however, pre-incubation of the cells with D609, a specific inhibitors of phosphatidylcholine-specific phospholipase C completely abolished the induction effect. These results clearly demonstrate that phosphatidylcholine-specific phospholipase C is a key molecule mediating insulin-induced enhancement of hIL-6 expression from the human cytomegalovirus promoter in Chinese hamster ovary cells and strongly suggest that it plays an important role in the insulin signaling pathways.Abbreviations CHO – Chinese hamster ovary; hCMV promoter – immediate early gene promoter of human cytomegalovirus; hIL-6 – human interleukin 6; PC-PLC-phosphatidylcholine-specific phospholipase C; PI-3 kinase – phosphoinositide 3 kinase; PKA – cAMP dependent protein kinase; PKC – protein kinase C.