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First published online 19 March 2009
doi: 10.1242/jcs.037747

Journal of Cell Science 122, 1258-1267 (2009)
Published by The Company of Biologists 2009
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p300-mediated acetylation of the Rothmund-Thomson-syndrome gene product RECQL4 regulates its subcellular localization

Tobias Dietschy1, Igor Shevelev1,2, Javier Pena-Diaz3, Daniela Hühn3, Sandra Kuenzle1, Raymond Mak1, Mohammad Fahad Miah1, Daniel Hess4, Monika Fey5, Michael O. Hottiger5, Pavel Janscak2,3 and Igor Stagljar1,*

1 Department of Biochemistry and Department of Molecular Genetics, Faculty of Medicine, Terrence Donnelly Centre for Cellular and Biomolecular Research (dCCBR), University of Toronto, 160 College Street, Toronto ON, Canada M5S 3E1
2 Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videnska 1083, 142 20 Prague, Czech Republic
3 Institute of Molecular Cancer Research, University of Zurich, Winterthurerstr. 190, CH-8057 Zurich, Switzerland
4 Protein Analysis Facility, Friedrich Miescher Institute, Maulbeerstr. 66, CH-4058 Basel, Switzerland
5 Institute of Veterinary Biochemistry and Molecular Biology, University of Zurich, Winterthurerstr. 190, CH-8057 Zurich, Switzerland


Figure 1
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  		Fig. 1. p300 acetylates RECQL4 in vivo and the two proteins form a stable complex. (A) Left panel: (His)6-Xpress-RECQL4 was ectopically expressed in HEK 293T cells along with FLAG-p300, myc-PCAF, HA-GCN5 or FLAG-HAT1 histone acetyltransferases (HATs). (His)6-Xpress-RECQL4 was then immunoprecipitated (IP) with Omni-probe antibody ({alpha}-Omni) and analyzed by western blot (upper panel). The same nitrocellulose membrane was stripped and re-probed with anti-acetyl-lysine ({alpha}-Ac-Lys) antibody (lower panel). Right panel: western blot analysis of overexpressed HATs (FLAG-p300, myc-PCAF, HA-GCN5 and FLAG-HAT1–FLAG-p46 heterodimer). Total protein extract (50 µg) was loaded in each lane. The same amount of extract from mock-transfected HEK 293T cells was analyzed as a negative control (–). (B) RECQL4 and p300 form a complex in human cells. Left panel: HEK 293T cells were transiently transfected with FLAG-p300 expression vector. Total cell extract derived from these cells was immunoprecipitated with anti-RECQL4 antibody ({alpha}-RECQL4) or control IgG (IgGctrl) and analyzed by SDS PAGE. One-tenth (100 µg) of the same total cell extract was used as input control (lane 1). Immunoprecipitated FLAG-p300 and RECQL4 were detected by western blotting using anti-FLAG ({alpha}-FLAG) and anti-RECQL4 ({alpha}-RECQL4) antibody, respectively (lane 3). Reciprocal co-immunoprecipitation is shown in the right panel: lane 4, input; lane 5, immunoprecipitation with the control IgG; lane 6, immunoprecipitation with anti-p300 antibody ({alpha}-p300) using total cell extracts derived from HEK 293T cells overexpressing (His)6-Xpress-RECQL4. (C) Binding of RECQL4 to p300 as a function of RECQL4 concentration. Increasing concentrations of RECQL4 (0–40 nM) were incubated at 37°C for one hour in wells of an ELISA plate that were pre-coated with the p300 protein (20 nM) and subsequently blocked with 3% BSA. After extensive washing, bound RECQL4 protein was detected as described in the Materials and Methods. Absorbance values were corrected by subtracting background values obtained with BSA-coated wells. Triangles represent the average of measurements from duplicate samples.

 

Figure 2
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  		Fig. 2. Mapping of interaction domains between p300 and RECQL4. (A) Schematic representation of RECQL4 and its deletion variants used in this study. (B) SDS-PAGE analysis of bacterially expressed and purified GST-RECQL4 fragments 1-4. Gel was stained with Coomassie blue. (C) GST pull-down assay showing binding of FLAG-p300 to bacterially expressed GST-RECQL4 fragments 1-4. GST-RECQL4 fragments bound to glutathione-Sepharose beads were incubated with whole cell extract (500 µg of total protein) derived from HEK 293T cells overexpressing FLAG-p300. Binding of p300 was analyzed by western blotting using anti-FLAG antibody ({alpha}-FLAG). Enhanced chemiluminescence (ECL) reagent was used for detection, and the film was exposed for 2 minutes. (D) Schematic representation of p300 and its deletion variants (p300-1 to p300-5). (E) SDS-PAGE analysis of bacterially expressed and purified GST-p300 fragments 1–5. (F) GST pull-down assay showing binding of RECQL4 to bacterially expressed GST-p300 fragments 1–5. GST-p300 fragments bound to glutathione-Sepharose beads were incubated with whole cell extract (1 mg of total protein) derived from HEK 293T cells overexpressing (His)6-Xpress-RECQL4. RECQL4 binding was analyzed by western blotting using Omni-probe antibody ({alpha}-Omni).

 

Figure 3
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  		Fig. 3. Mutation of lysine residues 376, 380, 382, 385 and 386 abrogates the acetylation of RECQL4 by p300 in vitro and in vivo. (A) Amino-acid sequence of RECQL4 NOS. Wild-type sequence is highlighted in yellow with lysine residues in red. Lysine to alanine (K->A) mutated NOS sequence and lysine to arginine (K->R) mutated NOS sequence of RECQL4 were generated by site-directed mutagenesis. Mutated residues are in red. (B) Left panel: Effect of mutation of lysine residues of RECQL4 NOS on in vivo acetylation of RECQL4. Using Omni-probe antibody ({alpha}-Omni), the wild-type (WT), K->A and K->R nucleolar localization signal mutants of (His)6-Xpress-RECQL4 were immunoprecipitated (IP) from extracts of HEK 293T cells co-transfected with p300 expression vector (+) or control vector (–) (upper panel). Acetylated (His)6-Xpress-RECQL4 was detected by western blot analysis using anti-acetyl lysine antibody ({alpha}-Ac-Lys) (lower panel). Right panel: Same as left panel, but the effects of single (K382R), double (K385, 386R) and triple (K376, 380, 382R) RECQL4 NOS mutations on in vivo acetylation of RECQL4 were tested. (C) Schematic representation of RECQL4 and its deletion variants used in this study (RECQL4-a to RECQL4-e). Numbers in italics indicate terminal amino acid positions. Solid black box, NOS; striped box, NOS with five lysine-to-alanine point mutations; green box, RecQ DNA helicase domain. (D) p300 purified from insect cells was incubated with [14C] acetyl coenzyme A and with purified GST-RECQL4 fragments or purified histone octamers. Left panel shows the Coomassie-blue-stained SDS-PAGE gel, and the right panel shows the autoradiogram of the same gel. Asterisks indicate protein bands with the predicted molecular mass of the corresponding purified GST-RECQL4 fragment.

 

Figure 4
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  		Fig. 4. Mutation of RECQL4 lysine residues Lys376, Lys380, Lys382, Lys385 and Lys386 to alanine (K->A), but not to arginine (K->R), causes relocalization of RECQL4 to the cytoplasm of human cells. (A) (His)6-Xpress-RECQL4 and the indicated (His)6-Xpress-RECQL4 K->R and K->A mutants were expressed in HeLa cells and visualized by indirect immunofluorescence using Omni-probe antibody ({alpha}-Omni) (red, centre column). The left column shows DAPI-stained nuclei (blue) and the right column shows the merged images. (B) The histograms show the percentage of transfected cells that show an equal distribution of RECQL4 between nucleus and cytoplasm (N=C), a prevalence of RECQL4 in the cytoplasm (N<C) or a prevalence of RECQL4 in the nucleus (N>C). The plotted data indicate the mean ± s.d. of two independent transfection experiments in which more than 200 transfected cells were analyzed each time. (C) Schematic representation of the RECQL4 NLS–β-galactosidase fusion constructs. Nucleotides encoding the RECQL4 amino-acid sequence 376-386 (wild type) and the indicated K->A mutant version of this sequence were C-terminally fused to the full-length cDNA of the E. coli lacZ gene and the fusion proteins were transiently expressed in HeLa cells. (D) β-galactosidase and the indicated fusion constructs (NLS β-Gal and NLS K->A β-Gal) were transiently transfected into HeLa cells and expressed proteins were visualized by indirect immunofluorescence using anti-β-galactosidase antibody (red, centre column). DAPI-staining (blue) is shown in the left column, and merged pictures (Merge) are shown in the right column. (E) Quantitative analysis as in B. Scale bars: 10 µm.

 

Figure 5
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  		Fig. 5. p300 activity-dependent accumulation of RECQL4 protein in the cytoplasm. (A) p300 and p300 {Delta}HAT, a catalytic dead mutant, localize to the nucleus in mammalian cells. FLAG-tagged p300 or FLAG-p300 {Delta}HAT proteins were transiently expressed in HeLa cells and visualized by indirect immunofluorescence using anti-FLAG antibody (green, centre column). DAPI-stained nuclei are shown in the left column (blue), and the merged pictures (Merge) are shown in the right column. (B,D) (His)6-Xpress-RECQL4 (B) or its K->R mutant (D) were co-expressed with FLAG-p300 or FLAG-p300 {Delta}HAT proteins in HeLa cells. RECQL4 proteins were visualized with Omni-probe antibody ({alpha}-Omni; red), and the p300 and p300 {Delta}HAT proteins were visualized with anti-FLAG antibody ({alpha}-FLAG; green). DAPI-staining (blue) shows nuclear DNA. Right-hand column show merged pictures (Merge). (C,E) Quantification of B and D. The histograms show the percentage of cells (out of transfected cells) that show an equal distribution of RECQL4 between nucleus and cytoplasm (N=C), a prevalence of RECQL4 in the cytoplasm (N<C) or a prevalence of RECQL4 in the nucleus (N>C). The plotted data represent the mean ± standard deviation of two independent transfection experiments in which more than 200 transfected cells were analyzed each time. Scale bars: 10 µm.

 

Figure 6
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  		Fig. 6. Histone deacetylase inhibitors trichostatin A and nicotinamide promote translocation of RECQL4 to the cytoplasm. (A) (His)6-Xpress-RECQL4 and (His)6-Xpress-RECQL4 K->R mutant were transiently overexpressed in HeLa cells. Trichostatin A (TSA) and nicotinamide (NA) were added 24 hours post-transfection and the cells left for an additional 30 hours. Cells were subsequently fixed and expressed proteins were visualized as in Fig. 4A. (B) Quantification of A. The plotted data indicate the mean ± standard deviation of two independent transfection experiments in which more than 200 transfected cells were analyzed each time. Scale bar: 10 µm.

 

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© The Company of Biologists Ltd 2009