The acidic C-terminal domain and A-box of HMGB-1 regulates p53-mediated transcription

Abstract

p53 function is modulated by several covalent and non-covalent modifiers. The architectural DNA- binding protein, High Mobility Group protein B-1 is a unique activator of p53. HMGB-1 protein is structured into two HMG-box domains, namely A-box and B-box, connected to a long highly acidic C-terminal domain. Here we report that both the C-terminal domain and A-box of HMGB-1 are critical for stimulation of p53-mediated DNA binding to its cognate site. Though deletion of these domains showed minimal effect in activation of p53-mediated transcription from the DNA template as compared to full-length HMGB-1, truncation of both the domains indeed showed significant reduction of transcriptional activation from the chromatin template as observed in DNA binding. Using transient transfection assays we showed that the C-terminal acidic domain and A-box of HMGB-1 are critical for the enhancement of the p53-mediated transactivation in vivo. Furthermore, the C-terminal domain and A-box deleted HMGB-1 could not activate p53-dependent apoptosis above the basal level. In conclusion, these results elucidate the role of acidic C-terminal domain and A-box of HMGB-1 in p53-mediated transcriptional activation and its further downstream effect.

Figure 1

Recombinant and native proteins used in the different experiments. (A) Diagrammatic representation of HMGB-1 and its truncated forms, HMGB-1ΔC and HMGB-1ΔA. (B) 200 ng of His₆-tagged HMGB-1FL (lane 1), 100 ng of HMGB-1ΔC (lane 2) and 200 ng of HMGB-1ΔA (lane 3) were analyzed by SDS–PAGE (18%). (C) The authenticity of these proteins was analyzed by western blot using HMGB-2 rabbit polyclonal antibody. HMG proteins were separated by 12% SDS–PAGE followed by western blot: HMGB-1FL (lane 1), HMGB-1ΔC (lane 2) and HMGB-1ΔA (lane 3). (D) FLAG-tagged full-length human p53 purified from E.coli and analyzed by SDS–PAGE (10%). The authenticity of the p53 protein was confirmed by western blot using p53 monoclonal antibody, DO1 (Oncogene). (E) Analysis of the recombinant Drosophila ACF complex by SDS–PAGE (8%) which was purified from FLAG-tagged ACF and ISWI baculovirus infected Sf21 cells. (F) Recombinant His₆-tagged mouse NAP1 purified from E.coli. (G) Native core histone purified from HeLa nuclear pellet and analyzed by SDS–PAGE (15%).

Figure 1

Recombinant and native proteins used in the different experiments. (A) Diagrammatic representation of HMGB-1 and its truncated forms, HMGB-1ΔC and HMGB-1ΔA. (B) 200 ng of His₆-tagged HMGB-1FL (lane 1), 100 ng of HMGB-1ΔC (lane 2) and 200 ng of HMGB-1ΔA (lane 3) were analyzed by SDS–PAGE (18%). (C) The authenticity of these proteins was analyzed by western blot using HMGB-2 rabbit polyclonal antibody. HMG proteins were separated by 12% SDS–PAGE followed by western blot: HMGB-1FL (lane 1), HMGB-1ΔC (lane 2) and HMGB-1ΔA (lane 3). (D) FLAG-tagged full-length human p53 purified from E.coli and analyzed by SDS–PAGE (10%). The authenticity of the p53 protein was confirmed by western blot using p53 monoclonal antibody, DO1 (Oncogene). (E) Analysis of the recombinant Drosophila ACF complex by SDS–PAGE (8%) which was purified from FLAG-tagged ACF and ISWI baculovirus infected Sf21 cells. (F) Recombinant His₆-tagged mouse NAP1 purified from E.coli. (G) Native core histone purified from HeLa nuclear pellet and analyzed by SDS–PAGE (15%).

Figure 2

The C-terminal acidic domain and A-box of HMGB-1 is essential to enhance sequence-specific DNA binding of p53. The effect of the C-terminal domain of HMGB-1 on p53-mediated DNA binding is shown. Human p53 (50 ng) was incubated with 3 ng of a ³²P-labeled p53 binding oligonucleotide in the absence (lane 1) or presence (lane 2) of 100, 200 and 300 ng of HMGB-1ΔA (lanes 3–5), HMGB-1ΔC (lanes 6–8) and HMGB-1FL (lanes 9–11). The ³²P-labeled probe was also incubated with only 100, 200 and 300 ng of HMGB-1ΔA (lanes 12–14), HMGB-1ΔC (lanes 15–17) and HMGB-1FL (lanes 18–20). Lane 1 contains only probe without any protein.

Figure 3

In vitro transcription from naked DNA template by p53 in the presence of HMGB-1FL and its truncated forms. (A) Schematic representation of the plasmid template (used for transcription) containing five p53 binding sites upstream of adenovirus major late core promoter (MLP) and G-less cassette. The transcription start site is represented as +1. (B) The newly constructed template P^(p53)₅^ML subjected to an in vitro transcription experiment using HeLa nuclear extract and without or with increasing concentrations of p53: lane 1, without p53; lane 2, 30 ng of p53; lane 3, 50 ng of p53; lane 4, 100 ng of p53; lane 5, 200 ng of p53. (C) Schematic representation of transcription protocol. (D) Effect of C-terminal domain on p53-mediated transcription. The DNA template (100 ng) was subjected to transcription according to the protocol in (C) with (lanes 2–8) or without (lane 1), 50 ng of p53 (lane 2) and the indicated amount of HMGB-1 and its truncated forms. Fifty, 100 and 200 ng of HMGB-1ΔC (lanes 3–5), and 50, 100 and 200 ng of HMGB-1FL (lanes 6–8) were added along with p53 (50 ng). (E) The effect of A-box of HMGB-1 on p53-mediated transcription from p^(p53)₅^ML naked DNA template. Transcription reactions were performed according to the protocol in (C), without p53 (lane 1) or with 50 ng of p53 (lane 2) and 50, 100 and 200 ng of HMGB-1ΔA (lanes 3–5), and 50, 100 and 200 ng of HMGB-1FL (lanes 6–8) in the presence of p53 (50 ng). (F) Effect of C-terminal domain on Gal4VP16-mediated transcription. The DNA template (100 ng) was subjected to transcription according to protocol (C). The transcription reaction was performed with Gal4VP16 (lanes 2–8) or without Gal4VP16 (lane 1). Fifty nanograms of Gal4VP16 (lane 2) were incubated with 50, 100 and 200 ng of HMGB-1ΔC (lanes 3–5), and 50, 100 and 200 ng of HMGB-1FL (lanes 6–8). (G) The effect of A-box of Gal4VP16-mediated transcription. The DNA template (100 ng) was subjected to transcription according to the protocol in (C). The transcription reaction was performed with Gal4VP16 (lanes 2–8) or without Gal4VP16 (lane 1). Fifty nanograms of Gal4VP16 (lane 2) were incubated with 50, 100 and 200 ng of HMGB-1ΔA (lanes 3–5), and 50, 100 and 200 ng of HMGB-1FL (lanes 6–8).

Figure 3

In vitro transcription from naked DNA template by p53 in the presence of HMGB-1FL and its truncated forms. (A) Schematic representation of the plasmid template (used for transcription) containing five p53 binding sites upstream of adenovirus major late core promoter (MLP) and G-less cassette. The transcription start site is represented as +1. (B) The newly constructed template P^(p53)₅^ML subjected to an in vitro transcription experiment using HeLa nuclear extract and without or with increasing concentrations of p53: lane 1, without p53; lane 2, 30 ng of p53; lane 3, 50 ng of p53; lane 4, 100 ng of p53; lane 5, 200 ng of p53. (C) Schematic representation of transcription protocol. (D) Effect of C-terminal domain on p53-mediated transcription. The DNA template (100 ng) was subjected to transcription according to the protocol in (C) with (lanes 2–8) or without (lane 1), 50 ng of p53 (lane 2) and the indicated amount of HMGB-1 and its truncated forms. Fifty, 100 and 200 ng of HMGB-1ΔC (lanes 3–5), and 50, 100 and 200 ng of HMGB-1FL (lanes 6–8) were added along with p53 (50 ng). (E) The effect of A-box of HMGB-1 on p53-mediated transcription from p^(p53)₅^ML naked DNA template. Transcription reactions were performed according to the protocol in (C), without p53 (lane 1) or with 50 ng of p53 (lane 2) and 50, 100 and 200 ng of HMGB-1ΔA (lanes 3–5), and 50, 100 and 200 ng of HMGB-1FL (lanes 6–8) in the presence of p53 (50 ng). (F) Effect of C-terminal domain on Gal4VP16-mediated transcription. The DNA template (100 ng) was subjected to transcription according to protocol (C). The transcription reaction was performed with Gal4VP16 (lanes 2–8) or without Gal4VP16 (lane 1). Fifty nanograms of Gal4VP16 (lane 2) were incubated with 50, 100 and 200 ng of HMGB-1ΔC (lanes 3–5), and 50, 100 and 200 ng of HMGB-1FL (lanes 6–8). (G) The effect of A-box of Gal4VP16-mediated transcription. The DNA template (100 ng) was subjected to transcription according to the protocol in (C). The transcription reaction was performed with Gal4VP16 (lanes 2–8) or without Gal4VP16 (lane 1). Fifty nanograms of Gal4VP16 (lane 2) were incubated with 50, 100 and 200 ng of HMGB-1ΔA (lanes 3–5), and 50, 100 and 200 ng of HMGB-1FL (lanes 6–8).

Figure 4

The C-terminal acidic domain and A-box of HMGB-1 is essential for p53-mediated transcriptional activation from chromatin template. (A) DNA supercoiling assays for in vitro assembled chromatin. Chromatin was assembled using the ATP regeneration system without ATP (lane 3) or with ATP (lane 4). Lane 1, supercoiled DNA used for chromatin assembly; lane 2, relaxed DNA after recombinant topoisomerase I treatment of the DNA of lane 1. (B) MNase digestion analysis of assembled chromatin. The chromatin was treated with varying concentrations of MNase for 6 min (lanes 2–5) and fixed concentrations of MNase with varying times of 5 (lane 6) and 10 min (lane 7) at room temperature. After deproteinization, the resulting DNA was resolved on a 1.25% agarose gel and stained with ethidium bromide. (C) Schematic diagram of in vitro transcription from chromatin template. (D) In vitro transcription from chromatin template. Freshly assembled chromatin template subjected to in vitro transcription as depicted in the protocol (C). Transcription reactions were performed without p53 (lane 1) or with 50 ng of p53 alone (lane 2) or in combination with 50 ng of HMGB-1ΔC (lane 3), 50 ng of HMGB-1ΔA (lane 4), 50 ng of HMGB-1FL (lane 5) and 100 ng of histone-free DNA treated in an assembly reaction was preincubated with 50 ng of p53 and 50 ng of HMGB-1FL and subjected to a transcription reaction (lane 6). (E) HMGB-1 and its truncated form cannot activate transcription of Gal4VP16 from the chromatin template. Freshly assembled chromatin template was subjected to in vitro transcription as depicted in the protocol (C). Transcription reactions were performed without Gal4VP16 (lane 1) or with 50 ng of Gal4VP16 alone (lane 2) or in combination with 50 ng of HMGB-1ΔC (lane 3), 50 ng of HMGB-1ΔA (lane 4), 50 ng of HMGB-1FL (lane 5) and 100 ng of histone-free DNA treated in an assembly reaction was preincubated with 50 ng of Gal4VP16 and 50 ng of HMGB-1FL and subjected to a transcription reaction (lane 6).

Figure 4

The C-terminal acidic domain and A-box of HMGB-1 is essential for p53-mediated transcriptional activation from chromatin template. (A) DNA supercoiling assays for in vitro assembled chromatin. Chromatin was assembled using the ATP regeneration system without ATP (lane 3) or with ATP (lane 4). Lane 1, supercoiled DNA used for chromatin assembly; lane 2, relaxed DNA after recombinant topoisomerase I treatment of the DNA of lane 1. (B) MNase digestion analysis of assembled chromatin. The chromatin was treated with varying concentrations of MNase for 6 min (lanes 2–5) and fixed concentrations of MNase with varying times of 5 (lane 6) and 10 min (lane 7) at room temperature. After deproteinization, the resulting DNA was resolved on a 1.25% agarose gel and stained with ethidium bromide. (C) Schematic diagram of in vitro transcription from chromatin template. (D) In vitro transcription from chromatin template. Freshly assembled chromatin template subjected to in vitro transcription as depicted in the protocol (C). Transcription reactions were performed without p53 (lane 1) or with 50 ng of p53 alone (lane 2) or in combination with 50 ng of HMGB-1ΔC (lane 3), 50 ng of HMGB-1ΔA (lane 4), 50 ng of HMGB-1FL (lane 5) and 100 ng of histone-free DNA treated in an assembly reaction was preincubated with 50 ng of p53 and 50 ng of HMGB-1FL and subjected to a transcription reaction (lane 6). (E) HMGB-1 and its truncated form cannot activate transcription of Gal4VP16 from the chromatin template. Freshly assembled chromatin template was subjected to in vitro transcription as depicted in the protocol (C). Transcription reactions were performed without Gal4VP16 (lane 1) or with 50 ng of Gal4VP16 alone (lane 2) or in combination with 50 ng of HMGB-1ΔC (lane 3), 50 ng of HMGB-1ΔA (lane 4), 50 ng of HMGB-1FL (lane 5) and 100 ng of histone-free DNA treated in an assembly reaction was preincubated with 50 ng of Gal4VP16 and 50 ng of HMGB-1FL and subjected to a transcription reaction (lane 6).

Figure 4

The C-terminal acidic domain and A-box of HMGB-1 is essential for p53-mediated transcriptional activation from chromatin template. (A) DNA supercoiling assays for in vitro assembled chromatin. Chromatin was assembled using the ATP regeneration system without ATP (lane 3) or with ATP (lane 4). Lane 1, supercoiled DNA used for chromatin assembly; lane 2, relaxed DNA after recombinant topoisomerase I treatment of the DNA of lane 1. (B) MNase digestion analysis of assembled chromatin. The chromatin was treated with varying concentrations of MNase for 6 min (lanes 2–5) and fixed concentrations of MNase with varying times of 5 (lane 6) and 10 min (lane 7) at room temperature. After deproteinization, the resulting DNA was resolved on a 1.25% agarose gel and stained with ethidium bromide. (C) Schematic diagram of in vitro transcription from chromatin template. (D) In vitro transcription from chromatin template. Freshly assembled chromatin template subjected to in vitro transcription as depicted in the protocol (C). Transcription reactions were performed without p53 (lane 1) or with 50 ng of p53 alone (lane 2) or in combination with 50 ng of HMGB-1ΔC (lane 3), 50 ng of HMGB-1ΔA (lane 4), 50 ng of HMGB-1FL (lane 5) and 100 ng of histone-free DNA treated in an assembly reaction was preincubated with 50 ng of p53 and 50 ng of HMGB-1FL and subjected to a transcription reaction (lane 6). (E) HMGB-1 and its truncated form cannot activate transcription of Gal4VP16 from the chromatin template. Freshly assembled chromatin template was subjected to in vitro transcription as depicted in the protocol (C). Transcription reactions were performed without Gal4VP16 (lane 1) or with 50 ng of Gal4VP16 alone (lane 2) or in combination with 50 ng of HMGB-1ΔC (lane 3), 50 ng of HMGB-1ΔA (lane 4), 50 ng of HMGB-1FL (lane 5) and 100 ng of histone-free DNA treated in an assembly reaction was preincubated with 50 ng of Gal4VP16 and 50 ng of HMGB-1FL and subjected to a transcription reaction (lane 6).

Figure 5

Acidic C-terminal domain and A-box of HMGB-1 specifically stimulates transactivation by p53. (A) H1299 cells were transiently transfected with p53 (250 ng) and HMGB-1FL, HMGB-1ΔC and HMGB-1ΔA (2.5 µg) either alone or in combination. The amount of transfected DNA was normalized with equivalent amounts of control parental vectors. The reporter constructs used was PG13Luc (1 µg). CMV-βgal (1 µg) was used as internal control for all the transfection experiments. Relative luciferase activity is plotted (y-axis) after normalization of luciferase activity with β-galactosidase activity. The relative luciferase activity is an average of three experiments. (B) H1299 cells were transiently transfected with Gal4VP16 (100 ng) and HMGB-1FL, HMGB-1ΔC and HMGB-1ΔA (1 µg) either alone or in combination. The amount of transfected DNA was normalized with the equivalent amount of control parental vectors. The reporter construct used was G10Luc (500 ng). CMV-βgal (500 ng) was used as an internal control for all the transfection experiments. Relative luciferase activity is plotted (y-axis) after normalization of luciferase activity with β-galactosidase activity. The relative luciferase activity is an average of three experiments. VP16, Gal4 Vp16.

Figure 5

Acidic C-terminal domain and A-box of HMGB-1 specifically stimulates transactivation by p53. (A) H1299 cells were transiently transfected with p53 (250 ng) and HMGB-1FL, HMGB-1ΔC and HMGB-1ΔA (2.5 µg) either alone or in combination. The amount of transfected DNA was normalized with equivalent amounts of control parental vectors. The reporter constructs used was PG13Luc (1 µg). CMV-βgal (1 µg) was used as internal control for all the transfection experiments. Relative luciferase activity is plotted (y-axis) after normalization of luciferase activity with β-galactosidase activity. The relative luciferase activity is an average of three experiments. (B) H1299 cells were transiently transfected with Gal4VP16 (100 ng) and HMGB-1FL, HMGB-1ΔC and HMGB-1ΔA (1 µg) either alone or in combination. The amount of transfected DNA was normalized with the equivalent amount of control parental vectors. The reporter construct used was G10Luc (500 ng). CMV-βgal (500 ng) was used as an internal control for all the transfection experiments. Relative luciferase activity is plotted (y-axis) after normalization of luciferase activity with β-galactosidase activity. The relative luciferase activity is an average of three experiments. VP16, Gal4 Vp16.

Figure 6

Effect of C-terminal domain and A-box of HMGB-1 in p53-mediated apoptosis. H1299 cells were transfected with 2.5 µg of p53 or 5 µg of HMGB-1FL, HMGB-1ΔC and HMGB-1ΔA either alone or in combination. Hoechst stain cells were observed under a fluorescence microscope using a blind approach. Apoptotic nuclei are indicated (arrows). The percentage of apoptotic nuclei refers to the average number of apoptotic cells present in 300 nuclei over three independent experiments (I).

Figure 6

Effect of C-terminal domain and A-box of HMGB-1 in p53-mediated apoptosis. H1299 cells were transfected with 2.5 µg of p53 or 5 µg of HMGB-1FL, HMGB-1ΔC and HMGB-1ΔA either alone or in combination. Hoechst stain cells were observed under a fluorescence microscope using a blind approach. Apoptotic nuclei are indicated (arrows). The percentage of apoptotic nuclei refers to the average number of apoptotic cells present in 300 nuclei over three independent experiments (I).

Figure 6

Effect of C-terminal domain and A-box of HMGB-1 in p53-mediated apoptosis. H1299 cells were transfected with 2.5 µg of p53 or 5 µg of HMGB-1FL, HMGB-1ΔC and HMGB-1ΔA either alone or in combination. Hoechst stain cells were observed under a fluorescence microscope using a blind approach. Apoptotic nuclei are indicated (arrows). The percentage of apoptotic nuclei refers to the average number of apoptotic cells present in 300 nuclei over three independent experiments (I).

Figure 6

Effect of C-terminal domain and A-box of HMGB-1 in p53-mediated apoptosis. H1299 cells were transfected with 2.5 µg of p53 or 5 µg of HMGB-1FL, HMGB-1ΔC and HMGB-1ΔA either alone or in combination. Hoechst stain cells were observed under a fluorescence microscope using a blind approach. Apoptotic nuclei are indicated (arrows). The percentage of apoptotic nuclei refers to the average number of apoptotic cells present in 300 nuclei over three independent experiments (I).