Importin-7 mediates nuclear trafficking of DNA in mammalian cells

Abstract

Eukaryotic cells have the ability to uptake and transport endogenous and exogenous DNA in their nuclei, however little is known about the specific pathways involved. Here we show that the nuclear transport receptor importin 7 (imp7) supports nuclear import of supercoiled plasmid DNA and human mitochondrial DNA in a Ran and energy-dependent way. The imp7-dependent pathway was specifically competed by excess DNA but not by excess of maltose-binding protein fused with the classical nuclear localizing signal (NLS) or the M9 peptides. Transport of DNA molecules complexed with poly-l-lysine was impaired in intact cells depleted of imp7, and DNA complexes remained localized in the cytoplasm. Poor DNA nuclear import in cells depleted of imp7 directly correlated with lower gene expression levels in these cells compared to controls. Inefficient nuclear import of transfected DNA induced greater upregulation of the interferon pathway, suggesting that rapid DNA nuclear import may prevent uncontrolled activation of the innate immune response. Our results provide evidence that imp7 is a non-redundant component of an intrinsic pathway in mammalian cells for efficient accumulation of exogenous and endogenous DNA in the nucleus, which may be critical for the exchange of genetic information between mitochondria and nuclear genomes and to control activation of the innate immune response.

Figure 1. Imp7 stimulates nuclear import of plasmid DNA

Nuclear import assay in permeabilized-HeLa cells in the presence of labelled plasmid DNA and: buffer; 1× energy mix (+Energy); 1× energy mix + 1× Ran mix (+Energy + Ran); 1× energy mix + 1× Ran mix + 1 µm imp7 (+Energy + Ran + imp7); 1 µm imp7 + Ran mix (+Ran + imp7); 1× energy mix + 1× Ran mix and 1 µm impβ (+Energy + Ran + impβ). Nuclear import was quantified by ImageJ software and the signal level in the buffer samples is given an arbitrary value of 100. Bars represent mean ± SD from 40–50 cells. Results are representative of four independent experiments.

Figure 2. Selective competition of plasmid DNA nuclear import

Nuclear import assays in permeabilized-HeLa cells in the presence of fluorescently labelled plasmid DNA (12.5 ng), 1 µm imp7, 1× energy and Ran mix and the indicated amounts of unlabelled plasmid DNA. Experiments were performed in parallel in the presence of 1 µm fluorescent MBP-M9 (M9) or GFP-NLS, 1 µm transportin or impα/β heterodimer, 1× energy and Ran mix and the indicated amounts of unlabelled plasmid DNA. Nuclear import was quantified using the Metamorph software. M9 import in the absence of competitor DNA is given an arbitrary value of 100. Values represent the average fluorescent intensity inside the nucleus relative to control (no DNA) ± SD of three independent experiments.

Figure 3. Imp7 induces nuclear import of human mtDNA

A) mtDNA was obtained from purified HeLa cells mitochondria after ultracentrifugation through a 4.5 m CsCl gradient and run on a 0.8% agarose gel. The band migrating with an apparent molecular weight of >23 kb was excised and eluted. MW, molecular weight markers; lanes 1 and 2, two different mtDNA preparations. B) Gel-fractionated mtDNA was analyzed by Southern blot with a COX I specific probe. Left panel, agarose gel after ethidium bromide staining; right panel, Southern blot. Lane 1, undigested mtDNA; lane 2, mtDNA cut with BamHI; lane 3, mtDNA cut with ClaI; lane 4, mtDNA cut with PvuII, lane 5, mtDNA cut with XhoI. These enzymes cut once in the mtDNA sequence . C) Purified mtDNA analyzed by PCR with primers specific for 28S rDNA or for COX I. MW, molecular weight markers, lanes 1–5, serial dilutions of genomic DNA from 30 to 0.3 ng amplified using 28S rDNA specific primers; lanes 6, no genomic DNA; lanes 7–10, serial dilutions of purified mtDNA from 30 to 1 ng; lane 11, mtDNA (1.25 ng) amplified with COX I specific primers; lane 12, no mtDNA. D) Nuclear import assay into permeabilized HeLa cells in the presence of 20 ng labelled mtDNA and: buffer (−E); 1× energy mix (E); 1× energy mix + 1× Ran mix (E + Ran); 1× energy mix + 1× Ran mix + 1 µm imp7 (imp7). Results are representative of two independent experiments. Scale bar = 10 µm.

Figure 4. DNA/PLL polyplex nuclear import is reduced in two stable Imp7 KD cell clones (CL2 and CL4) compared to control KD cells (DxR control) and imp7 back complemented cells (Back imp7)

Polyplexes (containing 2 µg DNA) were prepared at an equal surface charge at ratios of +1.6 (for SC- and OC-pDNA) and +5 for linear pDNA. A) Confocal microscopy analysis of polyplexes uptake at 1 h post-transfection. PLL is labelled green, DNA is labelled red, the nucleus is labelled blue and the cell cytoplasm is labelled grey. B) Polyplex confocal images were quantified in the periphery, cytosol and nucleus using IImageJ. The figure shows the mean and standard error (SE) of three independent experiments. C) Western blot with an anti-imp7 antibody and an anti-transportin 3 (Tnpo3) antibody (as a specificity control) on total cell extracts obtained from DxR HeLa cells, imp7 KD clone 2 (CL2) and clone 4 (CL4) and Back-imp7 cells used in the experiments.

Figure 5. The effect of Imp7 on polyplex gene expression (containing 20 µg DNA) after transfection into control (DxR), imp7 back complemented cells (Back imp7) and two stable Imp7 knockdown clones (imp7 KD CL2 and CL4)

β-Galactosidase gene expression was measured 48 h post-transfection. The bar graphs show the mean ± SE of three independent experiments (p < 0.05). SC-pDNA, polyplexes of supercoiled plasmid DNA; OC, polyplexes of open circular plasmid DNA; Linear pDNA, polyplexes of linear plasmid DNA.

Figure 6. The effect of Imp7 on innate immune responses to transfected DNA

The normalised levels of IFIT2 messenger RNA (relative to GAPDH; mean ± SE, n = 3) after transfection of DNA polyplexes (containing 2 µg pDNA) were measured in control (DxR), imp7 back complemented cells (Back imp7) and two independent stable Imp7 knockdown clones (imp7 KD CL2 and CL4). The figure shows the results of one representative experiment out of four independent experiments. Gene expression in untransfected cells, or in cells transfected with polylysine alone was undetectable.

Figure 7. The effect of imp7 on MHC class I expression

Control (DxR), back complemented (Back imp7) and two stable imp7 knockdown clones (imp7 KD CL2 and CL4) were transfected with 0.6 µg pHR plasmid or treated with 600 IU/mL IFN-β as shown. MHC class I expression was measured using flow cytometry. A) MHC induction is shown as % compared to mock transfected controls. Horizontal line shows 100% (i.e. no change relative to untransfected). Error bars show standard error of the mean of four independent experiments. Asterisk shows significant upregulation of MHC class I (**p < 0.01, *p < 0.05, Student's t-test ). B) A representative flow cytometry histogram plot showing shift in MHC class I expression profile in response to DNA in a imp7 knockdown clone (CL2) but not in a control (DxR) clone.