Chromatin boundary activities (BAs) were identified in Saccharomyces cerevisiae by genetic screening. Such BAs bound to sites flanking a reporter gene establish a nonsilenced domain within the silent mating-type locus HML. Interestingly, various proteins involved in nuclear-cytoplasmic traffic, such as exportins Cse1p, Mex67p, and Los1p, exhibit a robust BA. Genetic studies, immunolocalization, live imaging, and chromatin immunoprecipitation experiments show that these transport proteins block spreading of heterochromatin by physical tethering of the HML locus to the Nup2p receptor of the nuclear pore complex. Genetic deletion of NUP2 abolishes the BA of all transport proteins, while direct targeting of Nup2p to the bracketing DNA elements restores activity. The data demonstrate that physical tethering of genomic loci to the NPC can dramatically alter their epigenetic activity.

Mesenchymal stem cells (MSCs) facilitate functional recovery in numerous animal models of inflammatory and ischemic tissue-related diseases with a growing body of research suggesting that exosomes mediate many of these therapeutic effects. It remains unclear, however, which types of proteins are packaged into exosomes compared with the cells from which they are derived. In this study, using comprehensive proteomic analysis, we demonstrated that human primed MSCs secrete exosomes (pMEX) that are packaged with markedly higher fractions of specific protein subclasses compared with their cells of origin, indicating regulation of their contents. Notably, we found that pMEX are also packaged with substantially elevated levels of extracellular-associated proteins. Fibronectin was the most abundant protein detected, and data established that fibronectin mediates the mitogenic properties of pMEX. In addition, treatment of SHSY5Y cells with pMEX induced the secretion of growth factors known to possess mitogenic and neurotrophic properties. Taken together, our comprehensive analysis indicates that pMEX are packaged with specific protein subtypes, which may provide a molecular basis for their distinct functional properties.

Although it is well accepted that the constituents of the cellular microenvironment modulate a myriad of cellular processes, including cell morphology, cytoskeletal dynamics and uptake pathways, the underlying mechanism of how these pathways influence non-viral gene transfer have not been studied. Transgene expression is increased on fibronectin (Fn) coated surfaces as a consequence of increased proliferation, cell spreading and active engagement of clathrin endocytosis pathway. RhoGTPases mediate the crosstalk between the cell and Fn, and regulate cellular processes involving filamentous actin, in-response to cellular interaction with Fn. Here the role of RhoGTPases specifically Rho, Rac and Cdc42 in modulation of non-viral gene transfer in mouse mesenchymal stem (mMSCs) plated in a fibronectin microenvironment was studied. More than 90% decrease in transgene expression was observed after inactivation of RhoGTPases using difficile toxin B (TcdB) and C3 transferase. Expression of dominant negative RhoA (RhoAT19N), Rac1(Rac1T17N) and Cdc42 (Cdc42T17N) also significantly reduced polyplex uptake and transgene expression. Interactions of cells with Fn lead to activation of RhoGTPases. However, further activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes (RhoAQ63L, Rac1Q61L and Cdc42Q61L) did not further enhance transgene expression in mMSCs, when plated on Fn. In contrast, activation of RhoA, Rac1 and Cdc42 by expression of constitutively active genes for cells plated on collagen I, which by itself did not increase RhoGTPase activation, resulted in enhanced transgene expression. Our study shows that RhoGTPases regulate internalization and effective intracellular processing of polyplexes that results in efficient gene transfer.

Non-viral gene delivery is severely limited by its efficiency. Previous studies aiming to improve the efficiency of non-viral gene delivery have focused on improving the vector system, while the cellular microenvironment where the cells reside is only beginning to be exploited as a means to enhance gene transfer. In this study, the effect of different densities of extracellular matrix proteins namely collagen I (C I), vitronectin (Vt), laminin (Lm), collagen IV (C IV), fibronectin (Fn) and ECM gel (ECMg), and their combinations on gene transfer to mouse mesenchymal stem cells (mMSCs) was studied. Protein coatings that resulted in well spread cells such as Fn, ECMg and C IV, resulted in 14.6, 7 and 6.1 fold increase in transgene expression respectively when compared to uncoated surfaces. The transgene expression was up to 90% inhibited on C I coated surface, which led to less spread cells. Interestingly, the same trend was not observed for polyplex internalization, where protein coats that resulted in less spread cells, such as C I and Vt, resulted in higher polyplex internalization. Subsequently, decreased transgene expression corresponded with inhibited trafficking of the internalized complexes to the nucleus. The effect of combining multiple ECM proteins on non-viral gene transfer was also investigated. Surfaces coated with combinations including C I resulted in inhibition of transgene expression. Furthermore surfaces coated with combination of Vt, C IV and Lm resulted in a statistically similar enhancement in transgene expression as compared to fibronectin. For all ECM combinations analyzed, the extent of cell spreading mediated by the ECM protein had a 70% correlation with the extent of overall gene transfer observed. We believe that the cellular microenvironment can be engineered to promote efficient gene transfer.