Expressions of ASC-1. An RNA blot (Clontech) containing 2 μg of poly(A)⁺ mRNA from the indicated human tissues was hybridized with an ASC-1 probe under standard conditions (2). Equivalent loading was verified by hybridization with an actin cDNA probe (results not shown). S.I., small intestine; P.B.L., peripheral blood leukocyte. Size markers are as indicated.

(A) ASC-1 amino acid sequence. Two potential nuclear localization signals are underlined. Cysteine and histidine residues are indicated in boldface (those conserved with E1A [14] and VAC1 [51] are underlined). The positions of the deletions in ASCΔN and ASCΔNΔC are indicated by arrows. (B) Schematic diagram of ASC-1 and its deletion mutants. The putative zinc finger domain is as indicated. ASCΔN consists of the ASC-1 residues 125 to 581, and ASCΔNΔC includes only the putative zinc finger domain (residues 125 to 237). The zinc finger region is specifically deleted in ASCdn (i.e., the ASC-1 residues 165 to 216).

Autonomous transactivation function of ASC-1. (A) The yeast one-hybrid results. The indicated LexA-plasmids were transformed into a yeast strain containing an appropriate lacZ reporter gene, as described (29). The results are expressed as induction fold (n-fold) over the value obtained with LexA/−, which was given an arbitrary value of 1. The data are representative of at least two similar experiments and the standard deviations were less than 5%. (B) CV-1 cells were transfected with lacZ expression vector and increasing amounts of Gal4–ASC-1-expression vector, along with a reporter construct Gal4-TK-LUC, as described. Similar results were also obtained with HeLa cells and Gal4-ASCΔNΔC (results not shown). The results are expressed as n-fold over the value obtained with 100 ng of Gal4/N, which was given an arbitrary value of 1. The data are representative of at least two similar experiments, and the standard deviations were less than 5%. (C) In vitro interaction of ASC-1 with basal factors. Bacterially produced GST-TBP, GST-TFIIA, or GST alone was bound to a glutathione-agarose column and incubated with equivalent amounts of the indicated ³⁵S-labeled ASC-1, SRC-1, or IκBβ produced by in vitro translation, as described (2). Specifically bound proteins were released by glutathione and resolved by SDS-PAGE. Approximately 20% of the labeled proteins used in the binding reactions were loaded as inputs.

Expression of endogenous ASC-1 in HeLa cells. (A) HeLa cells were fractionated into nuclear (NE) and cytosolic (S-100) fractions as described (2) and were probed with indicated antibodies by Western analysis. α-RPB1 and α-SRC-1 are monoclonal antibodies against the RNA polymerase II largest subunit (8WG16) and SRC-1, respectively. (B) HeLa nuclear extracts were incubated with bacterially expressed GST-fusions to TR and ER or GST alone either in the presence or absence of ligand, as indicated. Specifically bound proteins were released by glutathione, resolved by SDS-PAGE, and probed with a rabbit polyclonal antibody against ASC-1 by Western analysis. Approximately 20% of the nuclear extracts used in the binding reactions were loaded as an input.

ASC-1 interacts with nuclear receptors. (A) The indicated B42 and LexA plasmids were transformed into a yeast strain containing an appropriate lacZ reporter gene, as described (29). TR-D and TR-E include the rat TRβ1 residues 164 to 265 (the hinge domain) and 260 to 444 (the LBD), respectively. Open and stippled boxes indicate the presence of B42 alone, whereas hatched and closed boxes indicate the presence of B42–ASC-1. Stippled and closed boxes include 1 μM T3 or 9-cis-RA. The results are expressed as induction fold (n-fold) over the value obtained with LexA/− and B42 alone, which was given an arbitrary value of 1. The data are representative of at least two similar experiments and the standard deviations were less than 5%. Similar results were also obtained with ASCΔN and ASCΔNΔC. (B) In vitro interaction of ASC-1 with nuclear receptors. Bacterially produced GST alone or GST fusions to RARα, TRβ, ERα, RXR, and RXRΔAF2 (57) were bound to a glutathione-agarose column and incubated with equivalent amounts of the indicated ³⁵S-labeled ASC-1 or SRC-1 produced by in vitro translation, as described (2). +, presence of 0.1 μM of each cognate ligand. Approximately 20% of the labeled proteins used in the binding reactions were loaded as inputs. (C) Ligand-dependent interaction of ASC-1 with TR in the presence of SMRT (20). GST alone or GST-TR bound to a glutathione-agarose column was incubated with ASC-1 or SMRT labeled by in vitro translation. +, presence of 0.1 μM T3. Approximately 20% of the labeled proteins used in the binding reactions were loaded as inputs.

ASC-1 interacts with SRC-1–CBP-p300. (A) The indicated B42 and LexA plasmids were transformed into a yeast strain containing an appropriate lacZ reporter gene, as described (29). SRC-C and SRC-D include the SRC-1 residues 568 to 779 and 759 to 1141, respectively. Open boxes indicate B42 alone, whereas stippled, hatched, and closed boxes indicate the presence of B42 fusions to ASC-1, ASCΔN, and ASCΔNΔC, respectively. The results are expressed as induction fold (n-fold) over the value obtained with LexA/− and B42 alone, which was given an arbitrary value of 1. The data are representative of at least two similar experiments and the standard deviations were less than 5%. (B) GST alone, GST-ASC-1, or GST-RXR bound to a glutathione-agarose column was incubated with SRC-C or SRC-D labeled by in vitro translation. +, presence of 0.1 μM 9-cis-RA. Approximately 20% of the labeled proteins used in the binding reactions were loaded as inputs. (C) The indicated B42 and LexA plasmids were transformed into a yeast strain containing an appropriate lacZ reporter gene, as described (29). CBP-C includes the CBP residues 1868 to 2441, whereas p300-N and p300-C include the p300 residues 1 to 117 and 2041 to 2157, respectively. Open boxes indicate B42 alone, whereas stippled, hatched, and closed boxes indicate the presence of B42 fusions to p300-N, p300-C, and CBP-C, respectively. The results are expressed as n-fold over the value obtained with LexA/− and B42 alone, which was given an arbitrary value of 1. The data are representative of at least two similar experiments and the standard deviations were less than 5%. (D) GST alone, GST-CBP-1, or GST-CBP-5 bound to a glutathione-agarose column was incubated with ASC-1 or p65 labeled by in vitro translation. Approximately 20% of the labeled proteins used in the binding reactions were loaded as inputs.

ASC-1 forms a ternary complex with RXR, CBP, and SRC-1. GST alone or GST-ASCΔNΔC bound to a glutathione-agarose column was incubated with CBP-C, RXR, or SRC-D labeled by in vitro translation. Increasing amounts of RXR (A) or SRC-D (B), in vitro translated in the absence of [³⁵S]methionine, was added to the binding reaction as a competitor, as indicated. Approximately 20% of the labeled proteins used in the binding reactions were loaded as inputs.

Association of ASC-1 with SRC-1, CBP, and liganded receptor in vivo. (A) Time-dependent accumulation of ASC-1 to cytoplasm in serum-starved cells. Rat-1 fibroblast cells were microinjected with HA-tagged ASC-1 plasmid (25 μg/ml) and fixed at indicated times during serum starvation, followed by indirect immunostaining with anti-HA antibody and FITC-rhodamine-conjugated antibodies as previously described (21). Serum (10%) or 0.1 μM 9-cis-RA was added to the media following 24 h of serum starvation and was observed 24 h later by indirect immunostaining, as indicated. The image was photographed with a Zeiss AxioplanII camera equipped with PIXERA. The picture is representative of approximately 50 injected cells which gave similar results. Similar results were also obtained with HeLa cells (results not shown). (B) Inhibition of cytoplasmic accumulation of ASC-1 by ligand, SRC-1, or CBP in serum-starved cells. Rat-1 fibroblast cells were microinjected with either HA-tagged ASC-1 (25 μg/ml) alone, HA-tagged ASC-1 plus SRC-1 (25 μg/ml each), or HA-tagged ASC-1 plus CBP (25 μg/ml each), were serum starved for 24 h either in the presence or absence of 0.1 μM 9-cis-RA as indicated, and were processed for immunostaining. Similar results were also obtained with HeLa cells (results not shown).

ASC-1 potentiates gene expressions mediated by nuclear receptors. HeLa cells were transfected with β-gal expression vector and increasing amounts of ASC-1-expression vector, either in the absence or presence of SRC-1 or p300 expression vector, along with indicated reporter constructs, as described. For specific activation of each reporter construct, 10 ng of RXR (A), 50 ng of RXRΔAF2 (B), 10 ng of ERα (C), 10 ng of TRβ (D), 10 ng of RARα (E), or 50 ng of Gal4-VP16 (F) was also added. Hatched boxes indicate the presence of 0.1 μM each cognate ligand. The results are expressed as induction fold (n-fold) over the value obtained in the absence of ligand and ASC-1, which was given an arbitrary value of 1. The data are representative of at least two similar experiments, and the standard deviations were less than 5%. Similar results were also obtained with CV-1 cells (results not shown).

Model of ASC-1 actions. Distinct putative coactivator complexes, each containing ASC-1, SRC-1, or CBP, exist in vivo (37 and unpublished results) and mediate transactivations by a number of distinct transcription factors. Each of these complexes recognizes a discrete part or subunit of target transcription factors and may cooperate with each other for efficient coactivation. Alternatively, these distinct coactivator complexes may form a larger supracomplex, as suggested by direct bindings among SRC-1, CBP, and ASC-1. Serum starvation may lead to enrichment of non-ASC-1-containing coactivator complexes within the nucleus, which may preferentially regulate genes turned on under serum-deprived conditions.