E2F factors rate controls the dual role of CDE/E2F composite element: a model of E2F-regulated gene expression in plant development

The promoters of several E2F-regulated genes identified in plants contain a variety of E2F motifs, notably a composite element consisting of a "CDE-like element" C/GGCGG on one strand, described as repressor in animals, associated with an E2F element on the complementary strand. This detailed study throughout plant development using ribonucleotide reductase promoters, allows us to propose a model, where E2F and composite elements play a dual role. Such regulation is mainly conditioned by the availability of E2F factors in tissues and during the cell cycle in tobacco.     

Genetic regulation of MUC1 expression by Helicobacter pylori in gastric cancer cells

Helicobacter pylori infection of the stomach is associated with the development of gastritis, peptic ulcers, and gastric adenocarcinomas, but the mechanisms are unknown. MUC1 is aberrantly overexpressed by more than 50% of stomach cancers, but its role in carcinogenesis remains to be defined. The current studies were undertaken to identify the genetic mechanisms regulating H. pylori-dependent MUC1 expression by gastric epithelial cells. Treatment of AGS cells with H. pylori increased MUC1 mRNA and protein levels, and augmented MUC1 gene promoter activity, compared with untreated cells. H. pylori increased binding of STAT3 and MUC1 itself to the MUC1 gene promoter within a region containing a STAT3 binding site, and decreased CpG methylation of the MUC1 promoter proximal to the STAT3 binding site, compared with untreated cells. These results suggest that H. pylori upregulates MUC1 expression in gastric cancer cells through STAT3 and CpG hypomethylation.     

Characterization of vertebrate cohesin complexes and their regulation in prophase

In eukaryotes, sister chromatids remain connected from the time of their synthesis until they are separated in anaphase. This cohesion depends on a complex of proteins called cohesins. In budding yeast, the anaphase-promoting complex (APC) pathway initiates anaphase by removing cohesins from chromosomes. In vertebrates, cohesins dissociate from chromosomes already in prophase. To study their mitotic regulation we have purified two 14S cohesin complexes from human cells. Both complexes contain SMC1, SMC3, SCC1, and either one of the yeast Scc3p orthologs SA1 and SA2. SA1 is also a subunit of 14S cohesin in Xenopus. These complexes interact with PDS5, a protein whose fungal orthologs have been implicated in chromosome cohesion, condensation, and recombination. The bulk of SA1- and SA2-containing complexes and PDS5 are chromatin-associated until they become soluble from prophase to telophase. Reconstitution of this process in mitotic Xenopus extracts shows that cohesin dissociation does neither depend on cyclin B proteolysis nor on the presence of the APC. Cohesins can also dissociate from chromatin in the absence of cyclin-dependent kinase 1 activity. These results suggest that vertebrate cohesins are regulated by a novel prophase pathway which is distinct from the APC pathway that controls cohesins in yeast.     

Estrogen regulation of trefoil factor 1 expression by estrogen receptor alpha and Sp proteins

Estrogen-responsive genes in human breast cancer cells often have an estrogen response element (ERE) positioned next to an Sp1 binding site. In chromatin immunoprecipitation (ChIP) assays, we investigated the binding of estrogen receptor alpha (ER), Sp1, and Sp3 to the episomal and native estrogen-responsive trefoil factor 1 (TFF1; formerly pS2) promoter in MCF-7 breast cancer cells. Mutation of the Sp site upstream of the ERE reduced estrogen responsiveness and prevented binding of Sp1 and Sp3, but not ER to the episomal promoter. In the absence of estradiol (E2), Sp1, Sp3, histone deacetylase 1 (HDAC), and HDAC2, and low levels of acetylated H3 and H4 are associated with the native promoter, with the histones being engaged in dynamic reversible acetylation. Following E2 addition, levels of ER and acetylated H3 and H4 bound to the native promoter increases. There is clearance of Sp1, but not of Sp3, from the promoter while HDAC1 and HDAC2 remain bound. These data are consistent with a model in which Sp1 or Sp3 aid in recruitment of HDACs and histone acetyltransferases (HATs) to mediate dynamic acetylation of histones associated with the TFF1 promoter, which is in a state of readiness to respond to events occurring following the addition of estrogen.     

Endoreduplication and activation of the anaphase-promoting complex during symbiotic cell development

Postembryonic development of plant organs requires a constant interplay between the cell cycle and the developmental programs. Upon endo- and exogenous signals, plant cells can enter, exit or modify the cell cycle. Alteration of mitotic cycles to endoreduplication cycles, where the genome is duplicated without mitosis, is common in plants and may play a role in cell differentiation. The switch from the mitotic to endocycles is regulated by Ccs52A, a plant orthologue of the yeast and animal Cdhl proteins, acting as substrate-specific activator of the anaphase-promoting complex E3 ubiquitin ligase. Here, several aspects of endoreduplication are discussed with special attention on nitrogen-fixing nodule development where endoreduplication is an integral part of symbiotic cell differentiation.     

ESPRG3多克隆抗体制备及初步功能研究

ESPRG3基因是一个与干细胞功能相关的基因,mESPRG3和hESPRG3基因被构建到原核及真核表达载体进行表达,原核表达蛋白用于制备多克隆抗体开展ESPRG3基因功能研究.真核表达载体EGFP被用于亚细胞定位.构建的mESPRG3基因原核及hESPRG3真核表达载体,成功进行了表达.利用IPTG诱导表达ESPRG3蛋白制备了多克隆抗体,免疫印迹和免疫组化结果表明制备的抗体具有特异性;表达的蛋白检测到体外具有结合DNA的能力.染色质免疫沉淀联合芯片技术检测到ESPRG3可以特异性结合到染色体上,且这些结合位点与Alu、及绝缘子序列具有关联性,可能通过这些位点调控胚胎干细胞的功能.     

Neuron-restrictive silencer factor functions to suppress Sp1-mediated transactivation of human secretin receptor gene

In the present study, a functional neuron restrictive silencer element (NRSE) was initially identified in the 5′ flanking region (− 83 to − 67, relative to ATG) of human secretin receptor (hSCTR) gene by promoter assays coupled with scanning mutation analyses. The interaction of neuron restrictive silencer factor (NRSF) with this motif was later indicated via gel mobility shift and ChIP assays. The silencing activity of NRSF was confirmed by over-expression and also by shRNA knock-down of endogenous NRSF. These studies showed an inverse relationship between the expression levels of NRSF and hSCTR in the cells. As hSCTR gene was previously shown to be controlled by two GC-boxes which are regulated by the ratio of Sp1 to Sp3, in the present study, the functional interactions of NRSF and Sp proteins to regulate hSCTR gene was investigated. By co-immunoprecipitation assays, we found that NRSF could be co-precipitated with Sp1 as well as Sp3 in PANC-1 cells. Interestingly, co-expressions of these factors showed that NRSF could suppress Sp1-mediated, but not Sp3-mediated, transactivation of hSCTR. Taken together, we propose here that the down-regulatory effects of NRSF on hSCTR gene expression are mediated via its suppression on Sp1-mediated transactivation.