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| Nuclear targeting
of plasmids and protein-DNA complexes |
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My laboratory
studies the mechanisms and applications of plasmid and DNA-binding
protein nuclear localization. Our long term goals are to
develop gene therapy approaches to the treatment of a variety
of human diseases by focusing on the development of novel
non-viral intra- and extracellular delivery methods. Our
main emphasis is in the area of vascular gene delivery and
function. Perhaps the major problem hindering gene therapy
is the inefficiency of gene transfer to slowly and non-dividing
cells. While many aspects of non-viral vector design are
being addressed, one critical area that has not received
adequate attention is the nuclear import of vector DNA. Clearly,
without the translocation of plasmid DNA into the nucleus,
no gene expression, or "gene therapy" can take
place. My laboratory continues to identify and characterize
novel DNA sequences to promote nuclear import of non-viral
vectors, both in cultured cells and in vivo.
Recent work from our laboratory has begun to address the
nuclear targeting and entry of plasmid DNA. Using cultured
cells, we have shown that plasmids are able to enter the
nuclei of cells in the absence of cell division and its accompanying
nuclear envelope breakdown. Assays used to follow the movement
of DNA include in situ hybridization, reporter gene expression,
and GFP-, YFP-, and BFP- tagged proteins. As for all other
macromolecular exchange between the cytoplasm and nucleus,
DNA nuclear entry is mediated by the nuclear pore complex.
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| Furthermore,
we have demonstrated that portions of the 72 bp SV40
enhancer are absolutely necessary for the nuclear entry
of plasmid DNA in all eukaryotic cells tested to date;
plasmids not containing this sequence remain in the cytoplasm
until cell division, whereas plasmids containing the
enhancer migrate to the nucleus within several hours.
These results demonstrate that transport of DNA into
the nucleus is sequence-specific. This 72 bp DNA fragment
contains multiple binding sites for various general transcription
factors: (click
image for larger view) |
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Since transcription factors bind to specific DNA sequences
and contain nuclear localization signals (NLSs) for their
nuclear import, it is likely that these proteins coat the
DNA with NLSs, thereby allowing the DNA-protein complex to
utilize the NLS-mediated import machinery for nuclear entry:
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Interestingly,
the SV40 sequence appears to be somewhat unique among
viral enhancers: neither the strong promoter/enhancers
of CMV or RSV have similar DNA nuclear import activity.
Click left image for larger view
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| While much of our work
is carried out in microinjected and transfected cells,
we have also developed novel DNA labeling techniques
using triplex-forming peptide nucleic acid (PNA) clamps
and have adapted cell-free systems to study the nuclear
import of protein and protein-DNA complexes in real time.
We have shown using |
permeabilized
cells that whereas the importin and proteins
and RAN are sufficient to drive the nuclear import
of an NLS protein, nuclear extracts are also
required for sequence-specific plasmid import.
Based on these results, we have developed a model
for general DNA nuclear import:
Click
image on right for larger view
We are focusing on identifying the proteins
present in the nuclear extract needed for this
activity by subtractive (purification) and
additive (using recombinant transcription factors
known to bind the SV40 enhancer) approaches.
One additional interesting
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feature of plasmid
nuclear import that we have discovered is that import
is inhibited when transcription is arrested. We have
demonstrated that this inhibition is not due to lack
of transcription of the import substrate (i.e., the plasmid)
but manifests its effect at the level of the cell. This
is strikingly similar to the transcription mediated nuclear
import inhibition seen with certain nuclear shuttling
proteins including the mRNA binding protein hnRNP A1.
Thus, we are working to understand how transcription
regulates nuclear import of DNA and the shuttling of
nuclear proteins, especially transcription factors.
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Our
model predicts that by using
DNA elements containing binding
sites for transcription factors
expressed in unique cell types,
we should be able to create plasmids
that target to the nucleus in
a cell-specific manner. Using
the promoter from the smooth
muscle gamma actin (SMGA) gene
whose expression is limited to
smooth muscle cells, we have
created a series of reporter
plasmids that are expressed selectively
in smooth muscle cells: (click
left image for larger view) |
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Moreover,
when injected into the cytoplasm,
plasmids containing portions
of the SMGA promoter localize
to the nucleus of smooth muscle
cells, but remain cytoplasmic
in fibroblasts and endothelial
cells. In contrast, a similar
plasmid carrying the SV40 enhancer
is transported into the nuclei
of all cell types tested. Limited
nuclear import of the SMGA promoter-containing
plasmids could be achieved when
the smooth muscle specific transcription
factor SRF was expressed in stably
transfected non-smooth muscle
cells, supporting our model for
the nuclear import of plasmids.
Moreover, when binding sites
for SRF or another smooth muscle
transcription factor, Nkx3, are
mutated, import is abolished,
further implicating these proteins
in nuclear import of the plasmids.
In collaboration with Warren
Zimmer (Univ. South Alabama)
we are continuing to identify
the factors that bind to the
SMGA promoter to regulate smooth
muscle specific transcription
and nuclear import using gel
shift assays, yeast two-hybrid
library screens, transient transfection
assays, DNaseI footprinting,
and nuclear import assays. Finally,
we have demonstrated that these
smooth muscle specific nuclear
targeting sequences are also
able to promote increased gene
expression in liposome- and polycation-transfected
non-dividing cells in a cell-specific
manner, similar to their nuclear
import activity. These results
provide proof of principle for
the development of cell-specific
non-viral vectors for any desired
cell type. Current studies involve
expanding our repertoire of cell-specific
DNA nuclear targeting sequences
so that we may be able to target
genes selectively to any desired
cell or tissue type.
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