A TEAD1/p65 complex regulates the eutherian-conserved MnSOD intronic enhancer, eRNA transcription and the innate immune response.

Manganese superoxide dismutase (MnSOD), a critical anti-oxidant enzyme, detoxifies the mitochondrial-derived reactive oxygen species, superoxide, elicited through normal respiration or the inflammatory response. Proinflammatory stimuli induce MnSOD gene expression through a eutherian-conserved, intronic enhancer element. We identified two prototypic enhancer binding proteins, TEAD1 and p65, that when co-expressed induce MnSOD expression comparable to pro-inflammatory stimuli.

Multiplexed detection of ions and mRNA expression in single living cells.

In order to push forward into new areas of medical and biological research, new techniques must be developed that will enable a complex investigation into cellular processes. This involves investigating not only the different expression levels inside of a cell but also the ability to analyze how those expression levels are connected to one another. In order to accomplish this level of exploration, different types of analytes must be investigated simultaneously inside of single cells, thereby allowing their expression levels to be directly compared.

Stochasticity of manganese superoxide dismutase mRNA expression in breast carcinoma cells by molecular beacon imaging.

Visual and quantitative monitoring of cell-to-cell variation in the expression of manganese superoxide dismutase (MnSOD) mRNA by using novel ratiometric imaging with molecular beacons (MB) reveals a distinct change in patterns following induction of human breast-carcinoma cells with lipopolysaccharide. Interestingly, the pattern of cell-to-cell variation in a cell line stably transfected with a plasmid bearing a cDNA clone of MnSOD and overproducing the enzyme is significantly different from the pattern associated with MnSOD induction. The levels and the patterns of cell-population heterogeneity for beta-actin mRNA expression do not show distinct changes either following induction or in stably transfected cells.

Simultaneous monitoring of the expression of multiple genes inside of single breast carcinoma cells.

Monitoring gene expression is at the center of research for a wide variety of medical, biological, and biotechnological applications. Currently no method exists for true multiple gene expression monitoring inside of a single living cell that allows for the gene expression profile of the cell to be directly compared with another single living cell. By microinjecting multiple molecular beacons with different fluorophores inside of single breast carcinoma cells and monitoring with advanced fluorescent microscopy, the expression of multiple genes can be simultaneously monitored inside single living cells.

PIN*POINT analysis on the endogenous MnSOD promoter: specific demonstration of Sp1 binding in vivo.

Manganese superoxide dismutase (MnSOD) is a critical antioxidant enzyme that protects against superoxide anion generated as a consequence of normal cellular respiration, as well as during the inflammatory response. By employing dimethyl sulfate in vivo footprinting, we have previously identified ten basal protein binding sites within the MnSOD promoter. On the basis of consensus sequence comparison and in vitro footprinting data, one would predict that Sp1 might occupy five of these binding sites.