Chee Kai Chan

Mutual exclusivity of DNA binding and nuclear localization signal recognition by the yeast transcription factor GAL4: implications for nonviral DNA delivery

A novel approach to nonviral DNA delivery is the use of combinations of DNA-binding proteins such as the yeast transcriptional activator GAL4 and plasmid DNA containing the specific binding sequence of the DNA-binding protein inserted within it, in addition to the gene of interest to be transferred into target cells. The amino terminal 147 amino acids of GAL4 contain a DNA-binding domain that has been shown to bind specifically to a 17 bp nucleotide recognition sequence, while the amino terminal 74 amino acids have been shown to be sufficient to target large heterologous proteins to the nucleus. Although it has been previously exploited as a gene transfer vehicle, the exact relationship between GAL4’s DNA binding and nuclear targeting activities has not been investigated. Using gel mobility shift assays and ELISA-based binding assays, this study examines this issue directly, establishing the mutual exclusivity of the DNA-binding and nuclear targeting activi- ties of GAL4. We demonstrate that GAL4(1–147) can specifically enhance transfection of plasmids containing the 17 bp recognition sequence. Interestingly, we found that the nuclear localization signal (NLS) of GAL4 is distinct from conventional NLSs, such as those of the SV40 large tumor antigen and bipartite NLSs, in that it is recognized exclusively by the nuclear pore targeting β-subunit of the NLS-receptor importin complex, rather than the α-subunit. Specific binding to DNA was blocked by β-subunit binding, while the converse was also true, making the GAL4-NLS novel in being regulated by DNA binding; this may play an important role in effecting release of GAL4 from the β-subunit following transport through the nuclear pore. This study encompasses the first direct analysis of NLS recognition/accessibility in vehicles for nonviral DNA transfer, with the results having relevance to the use of GAL4 and comparable DNA-binding proteins in such vehicles.

Plant importin alpha binds nuclear localization sequences with high affinity and can mediate nuclear import independent of importin beta.

Nuclear import of conventional nuclear localization sequence (NLS)-containing proteins initially involves recognition by the importin (IMP) α/β heterodimer, where IMPα binds the NLS and IMPβ targets the IMPα/NLS-containing protein complex to the nuclear pore. Here we examine IMPα from the plantArabidopsis thaliana (At-IMPα), which exhibits nuclear envelope localization typical of IMPβ rather than IMPα in other eukaryotic cell systems. We show that At-IMPα recognizes conventional NLSs of two different types with high affinity (K d of 5–10 nm), in contrast to mouse IMPα (m-IMPα), which exhibits much lower affinity (K d of 50–70 nm) and only achieves high affinity in the presence of m-IMPβ. Unlike m-IMPα, At-IMPα is thus a high affinity NLS receptor in the absence of IMPβ. Interestingly, At-IMPα was also able to bind with high affinity to NLSs recognized specifically by m-IMPβ and not m-IMPα, including that of the maize transcription factor Opaque-2. Reconstitution of nuclear import in vitroindicated that in the absence of exogenous IMPβ subunit but dependent on RanGDP and NTF2, At-IMPα was able to mediate nuclear accumulation to levels comparable with those mediated by m-IMPα/β. Neither m-IMPα nor -β was able to mediate nuclear import in the absence of the other subunit. At-IMPα’s novel NLS recognition and nuclear transport properties imply that plants may possess an IMPα-mediated nuclear import pathway independent of IMPβ in addition to that mediated by IMPα/β.

Signals mediating nuclear targeting and their regulation: application in drug delivery.

The recent progress with respect to understanding the signals mediating the transport of proteins in both directions through the NPC, and cellular proteins interacting with these signals to effect the transport process has made possible a number of advances in terms of the use of this information in a clinical setting. In particular, our knowledge of the mechanism of regulation of the process, and of how we may exploit the cellular transport machinery itself in a therapeutic situation, especially where there may be transport pathways specific to particular viruses, has advanced considerably. In this context, this review expounds current understanding of the signals conferring targeting to the nucleus, and their practical and potential use in delivering molecules of interest to the nucleus in a clinical context. It also deals with targeting signals conferring nuclear protein export/shuttling between nuclear and cytoplasmic compartments as well as with those conferring nuclear or cytoplasmic retention, and with the specific mechanisms regulating the activity of these signals, and in particular those regulating signal‐dependent nuclear protein import. Detailed understanding of the processes of signal‐mediated nuclear protein import/export and its regulation enables the considered application and optimization of approaches to target molecules of interest, such as plasmid DNA or toxic molecules, efficiently to the nucleus according to need in a clinical or research context, and enhance the expression or efficiency of their action, respectively. The use of nuclear targeting signals in this context is reviewed, and future possibilities in terms of the application of our growing understanding of nuclear transport and its regulation are discussed.

Using nuclear targeting signals to enhance non-viral gene transfer

Gene therapy involves the introduction of DNA‐encoding therapeutic gene products into appropriate cells of an affected individual. The limitations of the approach relate largely to the poor efficiency of the delivery of the therapeutic DNA to the nucleus. This review examines recent work in the area of non‐viral gene transfer, building on developments in the field of nuclear protein import and their application in the field of non‐viral gene transfer. In particular, advances in the area of enhancing DNA targeting to the nucleus are discussed, including the use of modular nuclear targeting signals recognised by the cellular nuclear import machinery and DNA condensing agents to facilitate passage through the nuclear pore. Optimising nuclear DNA delivery through these and other strategies should assist greatly in rendering gene therapy a viable and realistic possibility for treating disease.

Supramolecular structure and nuclear targeting efficiency determine the enhancement of transfection by modified polylysines

Polylysine (pLy) has been used as a DNA carrier in nonviral gene delivery systems because it forms complexes with plasmid DNA via charge interaction, and condenses it into a compact structure. We have recently shown that cross-linking nuclear localization sequences (NLSs) to pLy can enhance transfection by conferring specific recognition by the cellular nuclear import ‘receptor’, the NLS-binding importin α/β heterodimer. The present study examines and correlates for the first time the effect of the lysine/nucleotide (Ly/Nu) ratio on transfection, recognition by importin α/β, and structure as determined using electron microscopy (EM) and atomic force microscopy (AFM), for pLy–DNA complexes with and without NLSs or mutant versions thereof. Intriguingly, we observed two distinct peaks of transfection enhancement at Ly/Nu ratios of 0.4 and 4.0, attributable to specific NLS recognition by importins and DNA compaction, respectively. The results indicate a clear correlation between the pLy–DNA structure, importin α/β recognition, and gene transfer efficiency, thus underlining the importance of using pLy–DNA at the optimal Ly/Nu ratio.

Colony Stimulating Factor-1 Dependence of Paneth Cell Development in the Mouse Small Intestine

BACKGROUND & AIMS Paneth cells (PCs) secrete defensins and antimicrobial enzymes that contribute to innate immunity against pathogen infections within the mucosa of the small intestine. We examined the role of colony stimulating factor-1 (CSF-1) in PC development. METHODS CSF-1-deficient and CSF-1 receptor (CSF-1R)-deficient mice and administration of neutralizing anti-CSF-1R antibody were used to study the requirement of CSF-1 for the development of epithelial cells of the small intestine. CSF-1 transgenic reporter mice and mice that express only the membrane-spanning, cell-surface CSF-1 isoform were used to investigate regulation by systemic versus local CSF-1. RESULTS Mice deficient in CSF-1 or CSF-1R had greatly reduced numbers of mature PCs. PCs express the CSF-1R, and administration of anti-CSF-1R antibody to neonatal mice significantly reduced the number of PCs. Analysis of transgenic CSF-1 reporter mice showed that CSF-1-expressing cells are in close proximity to PCs. CSF-1/CSF-1R-deficient mice also had reduced numbers of the proliferating epithelial cell progenitors and lamina propria macrophages. Expression of the membrane-spanning, cell-surface CSF-1 isoform in CSF-1-deficient mice completely rescued the deficiencies of PCs, proliferating progenitors, and lamina propria macrophages. CONCLUSIONS These results indicate local regulation by CSF-1 of PC development, either directly, in a juxtacrine/paracrine manner, or indirectly, by lamina propria macrophages. Therefore, CSF-1R hyperstimulation could be involved in hyperproliferative disorders of the small intestine, such as Crohns disease and ulcerative colitis.

Enhancement of MSH receptor - and GAL4-mediated gene transfer by switching the nuclear import pathway

Efficient nuclear delivery of plasmid DNA represents a major barrier in nonviral gene transfer. One approach has been to use DNA-binding proteins such as GAL4 from yeast as DNA carriers with nuclear targeting properties. We recently showed, however, that GAL4 is inefficient in targeting DNA to the nucleus because its DNA-binding and nuclear targeting activities are mutually exclusive, which relates to the fact that GAL4 nuclear import occurs via a novel pathway. Here, we ‘switch’ this pathway to a more conventional one by adding a modified poly-lysine to which an optimized nuclear targeting signal, based on that of the SV40 large T-antigen, is linked. We also use a chimeric GAL4-α-melanocyte stimulating hormone (MSH) fusion protein to enable gene transfer to cells expressing the MSH receptor. Switching the nuclear import pathway of the transfecting complex significantly enhances receptor-mediated gene transfer through enabling interaction with desired components of the cellular nuclear import machinery. The present study represents the first demonstration that nuclear targeting signals can enhance receptor-mediated gene delivery, the approaches having important relevance to research and clinical applications, such as in generating transgenic or knock-out animals, or in gene therapy.

Synergy of importin α recognition and DNA binding by the yeast transcriptional activator GAL4

The N‐terminus of the yeast transcriptional activator GAL4 contains partially overlapping nuclear targeting and DNA binding functions. We have previously shown that GAL4 is recognised with high affinity by importin β and not by the conventional nuclear localisation sequence binding importin α subunit of the importin α/β heterodimer. The present study uses ELISA‐based binding and electrophoretic mobility shift assays to show that recognition of GAL4 by importin α can occur, but only when GAL4 is bound to its specific DNA recognition sequence. Intriguingly, binding by importin α enhances DNA binding on the part of GAL4, implying a synergistic co‐operation between these two functions. The results implicate a possible role for importin α in the nucleus additional to its established role in nuclear transport, as well as having implications for the use of GAL4 as a DNA carrier in gene therapy applications.

CSF-1 Receptor-Dependent Colon Development, Homeostasis and Inflammatory Stress Response

The colony stimulating factor-1 (CSF-1) receptor (CSF-1R) directly regulates the development of Paneth cells (PC) and influences proliferation and cell fate in the small intestine (SI). In the present study, we have examined the role of CSF-1 and the CSF-1R in the large intestine, which lacks PC, in the steady state and in response to acute inflammation induced by dextran sulfate sodium (DSS). As previously shown in mouse, immunohistochemical (IHC) analysis of CSF-1R expression showed that the receptor is baso-laterally expressed on epithelial cells of human colonic crypts, indicating that this expression pattern is shared between species. Colons from Csf1r null and Csf1op/op mice were isolated and sectioned for IHC identification of enterocytes, enteroendocrine cells, goblet cells and proliferating cells. Both Csf1r−/− and Csf1op/op mice were found to have colon defects in enterocytes and enteroendocrine cell fate, with excessive goblet cell staining and reduced cell proliferation. In addition, the gene expression profiles of the cell cycle genes, cyclinD1, c-myc, c-fos, and c-myb were suppressed in Csf1r−/− colonic crypt, compared with those of WT mice and the expression of the stem cell marker gene Lgr5 was markedly reduced. However, analysis of the proliferative responses of immortalized mouse colon epithelial cells (lines; Immorto-5 and YAMC) indicated that CSF-1R is not a major regulator of colonocyte proliferation and that its effects on proliferation are indirect. In an examination of the acute inflammatory response, Csf1r +/− male mice were protected from the adverse affects of DSS-induced colitis compared with WT mice, while Csf1r +/− female mice were significantly less protected. These data indicate that CSF-1R signaling plays an important role in colon homeostasis and stem cell gene expression but that the receptor exacerbates the response to inflammatory challenge in male mice.

Differential intracellular distribution of DNA complexed with polyethylenimine (PEI) and PEI-polyarginine PTD influences exogenous gene expression within live COS-7 cells.

BackgroundPolyethylenimine (PEI) is one of the most efficient and versatile non-viral vectors available for gene delivery. Despite many advantages over viral vectors, PEI is still limited by lower transfection efficiency compared to its viral counterparts. Considerable investigation is devoted to the modification of PEI to incorporate virus-like properties to improve its efficacy, including the incorporation of the protein transduction domain (PTD) polyarginine (Arg); itself demonstrated to facilitate membrane translocation of molecular cargo. There is, however, limited understanding of the underlying mechanisms of gene delivery facilitated by both PEI and PEI-bioconjugates such as PEI-polyarginine (PEI-Arg) within live cells, which once elucidated will provide valuable insights into the development of more efficient non-viral gene delivery vectors.MethodsPEI and PEI-Arg were investigated for their ability to facilitate DNA internalization and gene expression within live COS-7 cells, in terms of the percentage of cells transfected and the relative amount of gene expression per cell. Intracellular trafficking of vectors was investigated using fluorescent microscopy during the first 5 h post transfection. Finally, nocodazole and aphidicolin were used to investigate the role of microtubules and mitosis, respectively, and their impact on PEI and PEI-Arg mediated gene delivery and expression.ResultsPEI-Arg maintained a high cellular DNA uptake efficiency, and facilitated as much as 2-fold more DNA internalization compared to PEI alone. PEI, but not PEI-Arg, displayed microtubule-facilitated trafficking, and was found to accumulate within close proximity to the nucleus. Only PEI facilitated significant gene expression, whereas PEI-Arg conferred negligible expression. Finally, while not exclusively dependant, microtubule trafficking and, to a greater extent, mitotic events significantly contributed to PEI facilitated gene expression.ConclusionPEI polyplexes are trafficked by an indirect association with microtubules, following endosomal entrapment. PEI facilitated expression is significantly influenced by a mitotic event, which is increased by microtubule organization center (MTOC)-associated localization of PEI polyplexes. PEI-Arg, although enhancing DNA internalization per cell, did not improve gene expression, highlighting the importance of microtubule trafficking for PEI vectors and the impact of the Arg peptide to intracellular trafficking. This study emphasizes the importance of a holistic approach to investigate the mechanisms of novel gene delivery vectors.