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Structural and calorimetric studies demonstrate that the hepatocyte nuclear factor 1β (HNF1β) transcription factor is imported into the nucleus via a monopartite NLS sequence.
Wiedmann MM
,
Aibara S
,
Spring DR
,
Stewart M
,
Brenton JD
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The transcription factor hepatocyte nuclear factor 1β (HNF1β) is ubiquitously overexpressed in ovarian clear cell carcinoma (CCC) and is a potential therapeutic target. To explore potential approaches that block HNF1β transcription we have identified and characterised extensively the nuclear localisation signal (NLS) for HNF1β and its interactions with the nuclear protein import receptor, Importin-α. Pull-down assays demonstrated that the DNA binding domain of HNF1β interacted with a spectrum of Importin-α isoforms and deletion constructs tagged with eGFP confirmed that the HNF1β (229)KKMRRNR(235) sequence was essential for nuclear localisation. We further characterised the interaction between the NLS and Importin-α using complementary biophysical techniques and have determined the 2.4Å resolution crystal structure of the HNF1β NLS peptide bound to Importin-α. The functional, biochemical, and structural characterisation of the nuclear localisation signal present on HNF1β and its interaction with the nuclear import protein Importin-α provide the basis for the development of compounds targeting transcription factor HNF1β via its nuclear import pathway.
Fig. 1. (A) HNF1β protein expression levels in CCC cell lines OVISE, JHOC5, JHOC7, JHOC9, SKOV3 and a negative control HGSOC cell line, PEO1; (B) Data is the mean (n = 3, three biological replicates) and SEM. Expression levels were normalised to the housekeeping protein GAPDH. The expression levels observed with JHOC5 (P < 0.005, ∗∗∗) and OVISE (P < 0.05, ∗) cells were significantly higher than PEO1.
Fig. 2. eGFP imaging of PEO1 transduced lines with (a) eGFP-HNF1β (Lv103) showed primarily nuclear localisation of the GFP signal, whereas cells transduced with eGFP (Lv105) alone (b) showed both nuclear and cytoplasmic localisation. In cells transduced with eGFP-HNF1β (Lv103) in which the seven-residue NLS had been deleted (c, Mutant 8) the eGFP signal was mislocalized to the cytoplasm. The scale bar represents 80 μm. Images were taken on a Leica tandem confocal microscope using 20×, 40× and 60× objectives.
Fig. 3. (A) Stable complex formation between ΔIBB mouse Importin-α1 (50 kDa) and HNF1β (27 kDa) during size exclusion chromatography. Fractions were analysed by Coomassie stained SDS-PAGE (∗ marks the peak for the complex); (B) GST pull-down with liberated HNF1β showing pull-downs using different ΔIBB mouse Importin-α isoforms (red ∗).
Fig. 4. (A) ITC titration for the binding of the HNF1β NLS peptide to ΔIBB mImportin-α1. Fitting the data (with χ2/DoF = 2.9 × 104) gave a Kd of 13.6 ± 1.5 nM for 0.94 ± 0.13 sites, ΔH = −7.2 ± 1.6 kcal/mol, ΔS = −1.7 cal/mol/deg. (B) In pull-down assays, GST-HNF1β bound only the E mutant of ΔIBB Xenopus Importin-α1 (that impairs NLS binding at the minor site) Mr 50 kD, but not the D mutant (that impairs binding at the major site) or ED double mutant (see Giesecke and Stewart, 2010).
Fig. 5. (A) Overview of the 2.4 Å crystal structure of the ΔIBB mouse Importin-α1:HNF1βNLS complex. Two copies of the NLS peptide were observed where one was bound in the major site (blue) and another in the minor site (yellow). Each ARM repeat of mouse Importin-α1 has been labelled with alternating colours. (B) Final 2Fo-Fc electron density map around NLS peptide in major and minor binding sites on mouse Importin-α1 contoured at 1σ.
Fig. 6. Schematic illustration of the interactions of the HNF1βNLS peptide in the major (A) and minor (B) sites on ΔIBB mouse Importin-α1.
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