Synergism with germ line transcription factor Oct-4: viral oncoproteins share the ability to mimic a stem cell-specific activity

Abstract

Activation of transcription by Oct-4 from remote binding sites requires a cofactor that is restricted to embryonal stem cells. The adenovirus E1A protein can mimic the activity of this stem cell-specific factor and stimulates Oct-4 activity in differentiated cells. Here we characterize the Oct-4-E1A interaction and show that the E1A 289R protein harbors two independent Oct-4 binding sites, both of which specifically interact with the POU domain of Oct-4. Furthermore, we demonstrate that, like E1A, the human papillomavirus E7 oncoprotein also specifically binds to the Oct-4 POU domain. E7 and Oct-4 can form a complex both in vitro and in vivo. Expression of E7 in differentiated cells stimulates Oct-4-mediated transactivation from distal binding sites. Moreover, Oct-4, but not other Oct factors, is active when expressed in cells transformed by human papillomavirus. Our results suggest that different viruses have evolved oncoproteins that share the ability to target Oct-4 and to mimic a stem cell-specific activity.

FIG. 1

Oct-4 binds GST-E1A and GST-E7 in vitro. (a) Schematic representation of Oct-4 and E1A constructs used. (b) Equivalent amounts of GST (lanes 1 and 2) and GST-E1A (lanes 3 and 4) were used to bind 35S-labelled in vitro translated (IVT) Oct-4 (lanes 1 and 3) and Oct-1 (lanes 2 and 4) proteins, respectively, as indicated. Bound proteins were subjected to SDS-PAGE and autoradiography. Lane 5, 25% Oct-4 input; lane 6: 25% Oct-1 input. (c) Equivalent amounts of GST (lanes 1, 5, 9, 13, and 17), GST-E1A 1-90 (lanes 2, 6, 10, 14, and 18), GST E1A 127-204 (lanes 3, 7, 11, 15, and 19) and GST-E1A 140-204 (lanes 4, 8, 12, and 20) were used to bind IVT Oct-4 (lanes 1 to 4), 4N-POU4 (lanes 5 to 8), POU4-4C (lanes 9 to 12), POU4 (lanes 13 to 16) and Oct-1 (lanes 17 to 20). (d) Equivalent amounts of GST (lanes 1 to 5) and GST-E7 (lanes 6 to 10) were used to bind IVT Oct-4 (lanes 1 and 6), 4N-POU4 (lanes 2 and 7), POU4-4C (lanes 3 and 8), POU4 (lanes 4 and 9), and Oct-1 (lanes 5 and 10).

FIG. 1

Oct-4 binds GST-E1A and GST-E7 in vitro. (a) Schematic representation of Oct-4 and E1A constructs used. (b) Equivalent amounts of GST (lanes 1 and 2) and GST-E1A (lanes 3 and 4) were used to bind 35S-labelled in vitro translated (IVT) Oct-4 (lanes 1 and 3) and Oct-1 (lanes 2 and 4) proteins, respectively, as indicated. Bound proteins were subjected to SDS-PAGE and autoradiography. Lane 5, 25% Oct-4 input; lane 6: 25% Oct-1 input. (c) Equivalent amounts of GST (lanes 1, 5, 9, 13, and 17), GST-E1A 1-90 (lanes 2, 6, 10, 14, and 18), GST E1A 127-204 (lanes 3, 7, 11, 15, and 19) and GST-E1A 140-204 (lanes 4, 8, 12, and 20) were used to bind IVT Oct-4 (lanes 1 to 4), 4N-POU4 (lanes 5 to 8), POU4-4C (lanes 9 to 12), POU4 (lanes 13 to 16) and Oct-1 (lanes 17 to 20). (d) Equivalent amounts of GST (lanes 1 to 5) and GST-E7 (lanes 6 to 10) were used to bind IVT Oct-4 (lanes 1 and 6), 4N-POU4 (lanes 2 and 7), POU4-4C (lanes 3 and 8), POU4 (lanes 4 and 9), and Oct-1 (lanes 5 and 10).

FIG. 2

Oct-4 binds E7 in vivo. Whole-cell extracts (2 μg) were prepared from COS cells transiently transfected with 10 μg of pCMV-Oct-4 (A), cotransfected with 10 μg of pCMV-Oct-4 and 10 μg of pX-E7 (B), and cotransfected with 10 μg of pSG5-vErbA and 10 μg of pX-E7 (C). Extracts were immunoprecipitated with HPV16 E7 antibody. Immunoprecipitates [IP(α-E7)] and 20 μg of cell lysate were separated by SDS-PAGE, and proteins were transferred onto polyvinylpyrrolidone membrane. Membranes were cut into two parts to allow the simultaneous detection of E7 (lower panels), Oct-4 (panels A and B, upper portion), and vErbA (panel C, upper portion) by Western analysis. The three proteins with molecular weights of approximately 43,000 recognized by the Oct-4 antibody probably represent different phosphorylated forms of the protein (7, 18a). Ig, immunoglobulin; MW, molecular weight (10³).

FIG. 3

Oct-4 and E7 cooperate in transactivation from remote binding sites. (A) The 3T3-derived cell lines M/1 and E7/2 were cotransfected with 2.0 μg of p6W-109tkCAT and 3.0 μg of pCMV-Oct-4. Fold activation refers to the quotient of CAT activity in the presence and absence of expression vector. The mean values of two independent transfection experiments are plotted. (B) HeLa cells were cotransfected with 0.5 μg of p5Fd-109tkCAT or p6W-109tkCAT and increasing amounts of pCMV-Oct-4 as indicated. (C) EMSA of extracts of HeLa cells transiently transfected with increasing amounts of pCMV-Oct-4 as indicated. Arrows indicate the positions of complexes containing Oct-1 and Oct-4. mock, extract from mock transfected HeLa cells; probe, octamer motif containing fragment from immunoglobulin heavy chain gene enhancer (1W) (41).

FIG. 3

Oct-4 and E7 cooperate in transactivation from remote binding sites. (A) The 3T3-derived cell lines M/1 and E7/2 were cotransfected with 2.0 μg of p6W-109tkCAT and 3.0 μg of pCMV-Oct-4. Fold activation refers to the quotient of CAT activity in the presence and absence of expression vector. The mean values of two independent transfection experiments are plotted. (B) HeLa cells were cotransfected with 0.5 μg of p5Fd-109tkCAT or p6W-109tkCAT and increasing amounts of pCMV-Oct-4 as indicated. (C) EMSA of extracts of HeLa cells transiently transfected with increasing amounts of pCMV-Oct-4 as indicated. Arrows indicate the positions of complexes containing Oct-1 and Oct-4. mock, extract from mock transfected HeLa cells; probe, octamer motif containing fragment from immunoglobulin heavy chain gene enhancer (1W) (41).

FIG. 4

Cell lines were cotransfected with 0.5 μg of p6W-109tkCAT and increasing amounts of pCMV-Oct-4 (black bars) and pCMV-Oct-2A (white bars) as indicated. The value of the titration series representing maximum reporter activation is shown (see text).

FIG. 5

HeLa (A) and 293 (B) cells were cotransfected with 0.5 μg of p6W-109tkCAT and increasing amounts of pCMV-POU4, pCMV-4N-POU4, and pCMV-POU4-4C as indicated. Fold activation refers to the quotient of CAT activity in the presence and absence of expression vector. The mean values of two independent transfection experiments are plotted.