FIG. 1.

Ets-1 transactivation function requires CBP/p300. (A) The Ets-1- and c-Myb-responsive CD13/APN promoter is repressed by adenovirus 12S E1A protein, an inhibitor of CBP/p300. KG1a myeloblastic cells were transiently cotransfected with the CD13/APN promoter luciferase reporter (CD13/APN Luciferase), a secreted alkaline phosphatase gene reporter (MAP1-SEAP), and either expression plasmids for 12S E1A (12S E1A), the Δ2–36 12S E1A mutant incapable of binding CBP/p300 (Δ2–36 E1A), or empty expression vector (None). Luciferase values were normalized to SEAP activity derived from the internal transfection control reporter. 12S E1A has little effect on the MAP1-SEAP internal control reporter (data not shown). (B) E1A represses Ets-1-dependent transcription in KG1a cells from a CD13/APN promoter lacking c-Myb binding sites (mybmut luc). Mybmut luc reporter activity was assayed as in panel A. (C) Gal-Ets 2–440 is inhibited by 12S E1A in KG1a cells. KG1a cells were transiently transfected with G5B CAT reporter plasmid, RSV β-gal, and expression vectors for Gal4 DBD (Gal DBD), Gal4 DBD fused to the CBP/p300-independent-glutamine-rich activator from CREB aa 160 to 284 (Gal CREB 160–284), Gal DBD fused to murine Ets-1 aa 2 to 440 (Gal Ets 2–440), 12S E1A, or Δ2–36 E1A. CAT activity was normalized to β-gal activity derived from the internal transfection control RSV β-gal reporter plasmid. Empty expression vector gave similar results to those of the mutant Δ2–36 12S E1A (data not shown). (D) 12S E1A does not inhibit the expression of Gal-Ets 2–440 in KG1a cells. The cells were transiently transfected with the indicated expression vectors before undergoing metabolic labeling with [³⁵S]methionine; this was followed by whole-cell extract preparation and two sequential immunoprecipitations (IP) with anti-Ets-1(N- and C-terminal-specific) and anti-Gal4 DBD antibodies. (E) 12S E1A and Δ2–36 E1A are expressed at comparable levels in transiently transfected KG1a cells. The cells were labeled with [³⁵S]methionine before whole-cell extract preparation and immunoprecipitation with anti-E1A antibody. Arrows point to the respective E1A proteins. Molecular size markers are indicated (in kilodaltons). (F) p300 potentiates Ets-1 activity in transiently transfected F9 cells. The CD13/APN luciferase reporter was cotransfected with expression vectors for Ets-1 (+Ets-1) or empty vector (−Ets-1), p300 (CMVp300), or empty vector (CMV). Luciferase activity derived from the CD13/APN reporter was normalized to Renilla luciferase derived from the internal control reporter pRL-TK. (G) Full-length CBP potentiates Ets-1 activity in F9 cells. Transfections were performed as in panel F, except that full-length CBP (RSV CBP) was compared to a CBP expression vector (RSV CBP 1–1285) containing a nonsense mutation at CBP codon 1286. Results are means and standard errors (n ≥ 2).