Microtubule acetylation through HDAC6 inhibition results in increased transfection efficiency.
The success of viral and nonviral gene delivery relies on the ability of DNA-based vectors to traverse the cytoplasm and reach the nucleus. We, as well as other researchers, have shown that plasmids utilize the microtubule network and its associated motor proteins to traffic toward the nucleus. While disruption of microtubules with nocodazole was shown to greatly inhibit cytoplasmic plasmid trafficking, it did not abolish it. It has been demonstrated that a pool of stabilized post-translationally acetylated microtubules exists in cells, and that this acetylation may play a role in protein trafficking. In order to determine whether this modification could account for the residual DNA trafficking in nocodazole-treated cells, we inhibited or knocked down the levels of the tubulin deacetylase, histone deacetylase 6 (HDAC6), thereby generating higher levels of acetylated microtubules. Electroporation of plasmids into cells with inhibited or silenced HDAC6 resulted in increased gene transfer. This increased transfection efficiency was not because of increased transcriptional activity, but rather, because of increased cytoplasmic trafficking. When plasmids were cytoplasmically microinjected into HDAC6-deficient cells, they entered the nucleus within 5 minutes of injection, almost 10 times faster than in wild-type cells. Taken together, these results suggest that modulation of HDAC6 and the microtubule network can increase the efficiency of gene transfer.
Vaughan
Erin E
EE
Division of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
Geiger
R Christopher
RC
Miller
Aaron M
AM
Loh-Marley
Phoebe L
PL
Suzuki
Takayoshi
T
Miyata
Naoki
N
Dean
David A
DA
D000107:Acetylation; D001692:Biological Transport; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D005822:Genetic Vectors; D000073864:Histone Deacetylase 6; D056572:Histone Deacetylase Inhibitors; D006655:Histone Deacetylases / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008870:Microtubules / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D010957:Plasmids; D014162:Transfection
D056572:Histone Deacetylase Inhibitors; D008156:Luciferases; C433811:HDAC6 protein, human; D000073864:Histone Deacetylase 6; D006655:Histone Deacetylases; D015739:Nocodazole