MAGEA1 inhibits the expression of BORIS via increased promoter methylation

Melanoma-associated antigen A1 (MAGEA1) and BORIS (also known as CTCFL) are members of the cancer testis antigen (CTA) family. Their functions and expression-regulation mechanisms are not fully understood. In this study, we reveal new functions and regulatory mechanisms of MAGEA1 and BORIS in breast cancer cells, which we investigated in parental and genetically manipulated breast cancer cells via gene overexpression or siRNA-mediated downregulation. We identified the interaction between MAGEA1 and CTCF, which is required for the binding of MAGEA1 to the BORIS promoter and is critical for the recruitment of DNMT3a. A protein complex containing MAGEA1, CTCF and DNMT3a was formed before or after conjunction with the BORIS promoter. The binding of this complex to the BORIS promoter accounts for the hypermethylation and repression of BORIS expression, which results in cell death in the breast cancer cell lines tested. Multiple approaches were employed, including co-immunoprecipitation, glutathione S-transferase pull-down assay, co-localization and cell death analyses using annexin V-FITC/propidium iodide double-staining and caspase 3 activation assays, chromatin immunoprecipitation and bisulfite sequencing PCR assays for methylation. Our results have implications for the development of strategies in CTA-based immune therapeutics

Regulation of gene expression by FSP27 in white and brown adipose tissue

<p>Abstract</p> <p>Background</p> <p>Brown and white adipose tissues (BAT and WAT) play critical roles in controlling energy homeostasis and in the development of obesity and diabetes. The mouse Fat-Specific protein 27 (FSP27), a member of the cell death-inducing DFF45-like effector (CIDE) family, is expressed in both BAT and WAT and is associated with lipid droplets. Over-expression of FSP27 promotes lipid storage, whereas <it>FSP27 </it>deficient mice have improved insulin sensitivity and are resistant to diet-induced obesity. In addition, <it>FSP27</it>-deficient white adipocytes have reduced lipid storage, smaller lipid droplets, increased mitochondrial activity and a higher expression of several BAT-selective genes. To elucidate the molecular mechanism by which FSP27 controls lipid storage and gene expression in WAT and BAT, we systematically analyzed the gene expression profile of <it>FSP27-</it>deficient WAT by microarray analysis and compared the expression levels of a specific set of genes in WAT and BAT by semi-quantitative real-time PCR analysis.</p> <p>Results</p> <p>BAT-selective genes were significantly up-regulated, whereas WAT-selective genes were down-regulated in the WAT of <it>FSP27-</it>deficient mice. The expression of the BAT-selective genes was also dramatically up-regulated in the WAT of <it>leptin/FSP27 </it>double deficient mice. In addition, the expression levels of genes involved in multiple metabolic pathways, including oxidative phosphorylation, the TCA cycle, fatty acid synthesis and fatty acid oxidation, were increased in the <it>FSP27-</it>deficient WAT. In contrast, the expression levels for genes involved in extracellular matrix remodeling, the classic complement pathway and TGF-β signaling were down-regulated in the <it>FSP27-</it>deficient WAT. Most importantly, the expression levels of regulatory factors that determine BAT identity, such as CEBPα/β, PRDM16 and major components of the cAMP pathway, were markedly up-regulated in the WAT of <it>FSP27-</it>deficient mice. The expression levels of these regulatory factors were also up-regulated in <it>leptin/FSP27 </it>double deficient mice. Interestingly, distinct gene expression profiles were observed in the BAT of <it>FSP27-</it>deficient mice. Taken together, these data suggest that the WAT of <it>FSP27-</it>deficient mice have a gene expression profile similar to that of BAT.</p> <p>Conclusions</p> <p>FSP27 acts as a molecular determinant that controls gene expression for a diversity of metabolic and signaling pathways and, in particular, the expression of regulatory factors, including CEBPα/β, PRDM16 and components of the cAMP signaling pathway, that control the identity of WAT and BAT.</p

Burden of Disease in China - Contrasting Disease Burden Patterns of the General and the Migrant Workers Populations

Krämer A, Fischer F, Plaß D, et al. Burden of Disease in China - Contrasting Disease Burden Patterns of the General and the Migrant Workers Populations. Migration and Health in China - A joint project of United Nations Research Institute for Social Development Sun Yat-sen Center for Migrant Health Policy. Geneva: United Nations Research Institute for Social Development (UNRISD); 2014

Combination of FACS and Homologous Recombination for the Generation of Stable and High-Expression Engineered Cell Lines

<div><p>Traditionally, cell line generation requires several months and involves screening of over several hundred cell clones for high productivity before dozens are selected as candidate cell lines. Here, we have designed a new strategy for the generation of stable and high-expression cell lines by combining homologous recombination (HR) and fluorescence-activated cell sorting (FACS). High expression was indicated by the expression of secreted green fluorescent protein (SEGFP). Parental cell lines with the highest expression of SEGFP were then selected by FACS and identified by stability analysis. Consequently, HR vectors were constructed using the cassette for SEGFP as the HR region. After transfecting the HR vector, the cells with negative SEGFP expression were enriched by FACS. The complete exchange between SEGFP and target gene (TNFR-Fc) cassettes was demonstrated by DNA analysis. Compared with the traditional method, by integrating the cassette containing the gene of interest into the pre-selected site, the highest producing cells secreted a more than 8-fold higher titer of target protein. Hence, this new strategy can be applied to isolated stable cell lines with desirable expression of any gene of interest. The stable cell lines can rapidly produce proteins for researching protein structure and function and are even applicable in drug discovery.</p></div

HR strategy to generate cell lines with higher expression of the GOI.

<p>(A–E) Compared with the cell pools and clones derived by traditional selection, the growth of the cell pool and single cell clones selected by the HR strategy remained stable (A, C). In contrast, the Qp and total titer of TNFR-Fc was significantly increased in cell pools and clones generated by the HR method (B, D, E). Black columns are cell clones obtained by the HR strategy; light gray columns are cell clones selected by the traditional method (E). (F) Verification of authentic exchange events for the two cell clones derived by the HR strategy. PCR was performed on 1D1 and 1D1-S2 cell pools and all 8 single-clones with primer pairs as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091712#pone-0091712-g005" target="_blank">Fig. 5B</a>. Negative controls were CHO cells and H₂O. Positive controls were SEGFP and HR vectors. The band-shift in all cell clones indicated the SEGFP could be replaced by TNFR-Fc by site-integration exactly.</p

Construction and GOI expression of HR vectors.

<p>(A) Schematic diagram of HR vectors. Three HR vectors were constructed from the TNFR-Fc expression vector. Fragments of the SEGFP expression cassette with different lengths were used for HR in the upstream and downstream sequences of the TNFR-Fc expression cassette. (B) HR vectors were transiently transfected into CHO cells using the TNFR-Fc expression vector as the control. The titers and Qp of TNFR-Fc were measured and compared among these vectors.</p