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First published online August 29, 2005
doi: 10.1242/10.1242/jcs.02521

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The nuclear-envelope protein and transcriptional repressor LAP2ß interacts with HDAC3 at the nuclear periphery, and induces histone H4 deacetylation

Raz Somech^1,2,3,*, Sigal Shaklai^1,2,*, Orit Geller^1,2, Ninette Amariglio^1,2, Amos J. Simon^1,2, Gideon Rechavi^1,2, and Einav Nili Gal-Yam^1,2

¹ Sheba Cancer Research Center and the Institute of Hematology, The Chaim Sheba Medical Center, Tel-Hashomer 52621, Israel
² Sackler School of Medicine, Tel-Aviv University, Tel-Hashomer 52621, Israel
³ Dana Children's Hospital, Tel Aviv Sourasky Medical Center, Israel

View larger version (20K): [in a new window]   Fig. 1. LAP2ß represses transcriptional activity in a dose-dependent manner. (A) LAP2ß represses the activity of E2F transcription factors. The E2F reporter (0.5 µg) and pCMV-ß-gal (0.5 µg) were co-transfected with expression vectors for E2F1 (0.15 µg), E2F4 (0.15 µg), E2F5 (0.15 µg), DP1 (0.15 µg), DP2 (0.15 µg), DP3 (0.3 µg), and LAP2ß (1 µg) into U2OS cells as indicated. (B) LAP2ß represses transcriptional activity of E2F5-DP3, p53, NF-B and the constitutive RSV promoter in a dose-dependent manner, but not that of the constitutive CMV promoter. The specific reporters (0.1 µg) and pCMV-ß-gal (0.5 µg), together with expression vectors for E2F5 (0.15 µg) and DP3 (0.3 µg, i), p53 (0.1 µg, ii), p65 (0.5 µg, iii), constitutive RSV promoter (0.1 µg, iv), constitutive CMV promoter (0.1 µg, v), and increasing doses of LAP2ß (0.1-2.0 µg) were transfected into U2OS cells as indicated. For each factor, its known endogenous inhibitor was assayed [Rb (1.0 µg), MDM2 (0.5 µg) and IB (1 µg)]. (C) LAP2ß represses the activity of p65 upon TNF stimulation. U2OS cells were transfected with increasing doses of LAP2ß (0.1-2.0 µg) and IB (1.0 µg). 12 hours before harvesting, the cells were stimulated with TNF (200 units). For all reporter assays, the values shown represent the averages ± S.D. of duplicate (A) or triplicate (B,C) readings after the luciferase values were normalized to the ß-galactosidase values and compared with the mock control.

View larger version (42K): [in a new window]   Fig. 2. LAP2ß binds HDAC3 and both proteins are co-localized to the nuclear membrane. (A) Diagrams representing the LAP2ß and LAP2 protein structures. Numbers represent the corresponding amino acids. The LAP2ß-specific region (amino acids 219-328) that served as bait in the two-hybrid screen is delineated in bold stripes. (B) Schematic representation of the two-hybrid positive clones: 4.4 (0.6 kb) and 4.2 (0.8 kb), and the corresponding full-length cDNAs of HDAC3 and alternatively spliced HDAC3 (GenBank database accession numbers AF074881 and AF074882, respectively). The numbers above the two isoforms represent the corresponding amino acids and the numbers in italics below represent the corresponding nucleotides. The gray-striped area (amino acids 33-70 in both HDAC3 isoforms) represents the sequence common to both positive clones. (C) HDAC3 and LAP2ß bind in vitro. HDAC3 and HDAC1 (as a control) were expressed as GST fusion proteins in bacteria. LAP2ß was translated in vitro in the presence of [35S]-labeled methionine (10% of input, lane 1). Mixtures of [35S]-labeled LAP2ß with GST, GST-HDAC3 or GST-HDAC1 (lanes 1, 2 and 3, respectively) were incubated with glutathione-Sepharose beads. The bound proteins were eluted, separated by SDS-PAGE and identified by autoradiography (top) and Coomassie-Blue staining (bottom). (D) LAP2 does not bind HDAC3. HDAC3 was expressed as a GST fusion protein in bacteria. LAP2 was translated in vitro in the presence of [35S]-labeled methionine (lane 1). Mixtures of [35S]-labeled LAP2 with GST or GST-HDAC3 (lanes 1 and 2, respectively) were incubated with glutathione-Sepharose beads. The proteins were eluted, separated by SDS-PAGE and identified by autoradiography (top) and Coomassie-Blue staining (bottom). (E) Overexpressed HDAC3 and LAP2ß bind in vivo. U2OS cells were co-transfected with HDAC3 (1 µg) and LAP2ß (1 µg). Cell extracts were immunoprecipitated with anti-LAP2ß antibodies (lanes 3 and 6) followed by immunoblotting with anti-HDAC3 (lanes 1-3) or anti-LAP2ß (lanes 4-6) antibodies. Beads only were used as a control for non-specific binding (lanes 2 and 5). A whole-cell lysate was used as a control marker for HDAC3 and LAP2ß (lanes 1 and 4, respectively). (F) Endogenous binding of LAP2ß and HDAC3 upon PHA stimulation of PBMCs. 2x106 PBMCs were stimulated with 25 µg PHA. After 72 hours, cellular extracts were immunoprecipitated with anti-LAP2ß antibodies (lanes 3 and 6), followed by immunoblotting with anti-HDAC3 (lanes 1-3) or anti-LAP2ß (lanes 4-6) antibodies. Beads only were used as a control for non-specific binding (lanes 2 and 5). A whole-cell lysate was used as a control marker for HDAC3 and LAP2ß (lanes 1 and 4, respectively). (G) Co-localization of transfected HDAC3 and LAP2ß. U2OS cells were transfected with either HDAC3 alone (1.0 µg, i), HDAC3 and increasing doses of LAP2ß (0.1 µg, ii; 0.5 µg, iii; 1.0 µg, iv), HDAC1 alone (1.0 µg, v) or HDAC1 and LAP2ß (1.0 µg, vi). The cells were fixed and immunostained with anti-HDAC3, anti-HDAC1 and anti-LAP2ß antibodies, as indicated, and analysed by confocal microscopy.

View larger version (10K): [in a new window]   Fig. 3. TSA reduces the transcriptional repression induced by LAP2ß. U2OS cells were transfected with pCMV-ß-gal (0.5 µg) and expression vectors for E2F5 (0.15 µg)-DP3 (0.3 µg, i), p53 (0.1 µg, ii), p65 (0.5 µg, iii) together with their specific reporters, or constitutive RSV promoter (0.1 µg, iv) with or without LAP2ß (1.0 µg), as indicated. Cells were treated with TSA (0.1 mM) as indicated 12 hours before harvesting. The values shown represent the average ± S.D. of triplicate readings after the luciferase values were normalized to the ß-galactosidase values and compared with the mock control.

View larger version (34K): [in a new window]   Fig. 4. LAP2ß induces deacetylation of histone H4 in vitro and in vivo. (A) U2OS cells were transfected with either 1 µg LAP2ß (i) or HDAC1 (ii). Cells were fixed and immunostained with antibodies against acetylated histone H4, LAP2ß or HDAC1, as indicated, and analysed by confocal microscopy. (B) U2OS cells were transfected with LAP2ß (0.1 or 1.0 µg) or HDAC1 (1.0 µg), followed by treatment with 5 mM sodium butyrate. Cellular extracts were detected by western blot analysis, using antibodies against acetylated histone H4, LAP2ß and ß-actin, as indicated. (C) Sodium-butyrate-treated U2OS cells were transfected with 1 µg LAP2ß, HDAC3, LAP2ß and HDAC3, or p300, as indicated. Cellular extracts were prepared and protein amount was detected in each sample. Equal amounts of lysates were incubated with [3H]-acetylated histone H4 peptide. Released [3H]-acetate was measured in cpm using a ß-radiation counter. The experiment was repeated three times.

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