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. 1998 Mar;18(3):1303-11.
doi: 10.1128/MCB.18.3.1303.

The alpha chain of the nascent polypeptide-associated complex functions as a transcriptional coactivator

W V Yotov  1 , A Moreau, R St-Arnaud
Affiliations

The alpha chain of the nascent polypeptide-associated complex functions as a transcriptional coactivator

W V Yotov et al. Mol Cell Biol. 1998 Mar.

Abstract

We report the characterization of clone 1.9.2, a gene expressed in mineralizing osteoblasts. Remarkably, clone 1.9.2 is the murine homolog of the alpha chain of the nascent polypeptide-associated complex (alpha-NAC). Based on sequence similarities between alpha-NAC/1.9.2 and transcriptional regulatory proteins and the fact that the heterodimerization partner of alpha-NAC was identified as the transcription factor BTF3b (B. Wiedmann, H. Sakai, T. A. Davis, and M. Wiedmann, Nature 370:434-440, 1994), we investigated a putative role for alpha-NAC/ 1.9.2 in transcriptional control. The alpha-NAC/1.9.2 protein potentiated by 10-fold the activity of the chimeric activator GAL4/VP-16 in vivo. The potentiation was shown to be mediated at the level of gene transcription, because alpha-NAC/1.9.2 increased GAL4/VP-16-mediated mRNA synthesis without affecting the half-life of the GAL4/VP-16 fusion protein. Moreover, the interaction of alpha-NAC/1.9.2 with a transcriptionally defective mutant of GAL4/VP-16 was severely compromised. Specific protein-protein interactions between alpha-NAC/1.9.2 and GAL4/VP-16 were demonstrated by gel retardation, affinity chromatography, and protein blotting assays, while interactions with TATA box-binding protein (TBP) were detected by immunoprecipitation, affinity chromatography, and protein blotting assays. Based on these interactions that define the coactivator class of proteins, we conclude that the alapha-NAC/1.9.2 gene product functions as a transcriptional coactivator.

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Figures

FIG. 1
FIG. 1
α-NAC/1.9.2 localizes to the cytoplasm and nucleus in serum-deprived osteoblasts. MC3T3-E1 osteoblastic cells were stained with polyclonal antibodies raised against the fusion GST-1.9.2 protein. (A) Control staining with preimmune sera. (B to D) Staining for α-NAC/1.9.2. A strong signal was observed in the cytoplasm and the nucleus of serum-starved cells (B). Following stimulation with fetal calf serum for 4 h, α-NAC/1.9.2 localized mainly to the cytoplasm (C). Removal of serum after the 4-h stimulation provoked reentry of α-NAC/1.9.2 into the nucleus (D).
FIG. 2
FIG. 2
α-NAC/1.9.2 acts as a transcriptional coactivator. Transient transfection assays of the reporter plasmid 5Gal4-E1b-CAT (20) with expression vectors for GAL4/VP-16, GAL4/1.9.2, α-NAC/1.9.2, and BTF3b alone or in combination. (A) Relative fold induction levels of the CAT reporter gene in transfected cells. Vector represents the parental pBK-CMV vector in which the α-NAC/1.9.2 cDNA was subcloned to generate CMV-1.9.2. The expression level detected in cells transfected with the reporter alone was arbitrarily ascribed a value of 1. Results are expressed as mean fold induction ± standard error of four independent transfections. (B) Northern blot assays of total RNA from the transfected cells. The blot was first hybridized to a CAT probe (upper panel) and then was stripped and reprobed with an α-tubulin probe (lower panel). (C) Half-life of the GAL4/VP-16 protein in the presence or absence of α-NAC/1.9.2. P19 EC cells (24) were transfected with the GAL4/VP-16 expression vector alone or in combination with the α-NAC/1.9.2 expression vector. Cycloheximide was added to each dish, and cells were harvested at the indicated intervals. Samples were analyzed by Western blotting with an anti-GAL4/VP-16 antibody.
FIG. 3
FIG. 3
α-NAC/1.9.2 interacts with GAL4/VP-16. Results from gel mobility shift assays with purified recombinant proteins with a labeled 17-mer GAL4 probe are shown. GAL4(DBD-1–147) is a fusion protein between GST and the DBD (residues 1 to 147) of GAL4. GAL4(1–147)-VP-16 is the same as GAL4/VP-16. α-NAC denotes the purified full-length α-NAC/1.9.2 protein. Preimmune sera (lanes 7 and 8) and antibodies raised against α-NAC/1.9.2 (anti-α-NAC [lanes 8 and 10]) were added to some binding reaction mixtures.
FIG. 4
FIG. 4
Interactions between α-NAC/1.9.2 and wild-type and mutant GAL4/VP-16. (A) Affinity chromatography of crude lysates from bacteria transformed with an expression vector for wild-type GAL4/VP-16. Bound proteins were eluted with a step gradient of NaCl, and the immunoblot (lanes 1 to 9) was probed with an anti-GAL4/VP-16 antibody. (B) Affinity chromatography of crude lysates from bacteria expressing GAL4/VP16ΔFP442 eluted with a salt step gradient. In, input (1/100 of total material loaded on column); F.T., flowthrough. Molecular mass (kilodaltons) markers (M) are indicated to the left of each panel.
FIG. 5
FIG. 5
Interactions between α-NAC/1.9.2 and TBP. (A) Affinity chromatography of crude nuclear extracts eluted with a salt step gradient. The immunoblot (lanes 3 to 15) was probed with the anti-TBP antibody. Recombinant human TBP (rTBP) was migrated in lane 15. (B) Control affinity chromatography column loaded with the GST moiety alone. The same crude nuclear extract used in panel A was passed through the column. Immunoblotting was with the anti-TBP antibody. In, input (1/100 of total material loaded on column); F.T., flowthrough. Lanes 1 and 2 contained Coomassie blue-stained molecular mass (kilodaltons) markers (M) and input material, respectively.
FIG. 6
FIG. 6
Protein-protein interactions between α-NAC/1.9.2 and GAL4/VP-16 and between α-NAC/1.9.2 and TBP. Results of a far-Western protein blot assay show the interaction of biotinylated GST-1.9.2 with TBP (lanes 2 and 3) and GAL4/VP-16 (lane 4). The fusion protein GST-1.9.2(111–215) (Mr, 39,400) served as a negative control in this assay (lane 1). Asterisks point to the proper molecular size of GST-TBP (lane 2), cleaved TBP (lane 3), and GAL4/VP-16 (lane 4). Molecular mass (kilodaltons) markers are indicated in the left-hand lane.
FIG. 7
FIG. 7
α-NAC/1.9.2 is associated with TBP in vivo. Immunoprecipitation and immunoblotting assays demonstrating the specific interaction of α-NAC/1.9.2 with TBP in crude nuclear extracts. Immunoprecipitation reactions were performed with preimmune sera [lanes 3, 6, and 9]), anti-TBP antibodies (A [lanes 2, 5, and 8]), anti-αNAC antibodies (A [lanes 1, 4, and 7]), or anti-RNA polymerase II (pol. II) antibodies (B [lanes 1 to 4]). The immunocomplexes were then analyzed by immunoblotting with the antibodies indicated in the upper portion of each panel. TBP was specifically associated with the anti-αNAC immunocomplexes (A, lane 7). Similarly, α-NAC/1.9.2 was specifically associated with the anti-TBP immunocomplexes (A [lane 5]). M, molecular mass (kilodaltons) markers (M). IgG, immunoglobulin G.
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