Limits...
The adenovirus E1A oncoprotein N-terminal transcriptional repression domain enhances p300 autoacetylation and inhibits histone H3 Lys18 acetylation.

Zhao LJ, Loewenstein PM, Green M - Genes Cancer (2015)

Bottom Line: Additional acetylation of p300 in the presence of E1A 1-80 produces stronger inhibition of H3K18 acetylation.These findings indicate that autoacetylation of p300 greatly reduces its ability to acetylate H3K18.The results reported here combined with our previous findings suggest that E1A can repress transcription by multiple strategies, including altering the chromatin modifying activity of p300 and dissociating TFIID from the TATA box thus disrupting formation of the transcription pre-initiation complex [5, 6].

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Virology, Saint Louis University School of Medicine, Doisy research Center, St. Louis, Missouri.

ABSTRACT
Expression of the adenovirus E1A N-terminal transcription repression domain alone (E1A 1-80) represses transcription from specific promoters such as HER2 [1] and from reconstituted chromatin [2]. Significantly, E1A 1-80 can induce the death of human breast cancer cells over-expressing the HER2 oncogene [1] as well as other cancer cells. Here, we report that E1A 1-80 alone is sufficient to inhibit H3K18 acetylation in vivo and p300-mediated H3K18 acetylation in reconstituted chromatin. Of interest, hypoacetylation of H3K18 has been correlated with the survival of tumor cells and the poor prognosis of many cancers [3, 4]. E1A 1-80 enhances p300 autoacetylation and concurrently inhibits H3K18 acetylation in chromatin in a dose-dependent manner. Pre-acetylation of p300 by incubation with acetyl-CoA alone reduces p300's ability to acetylate H3K18 in chromatin. Additional acetylation of p300 in the presence of E1A 1-80 produces stronger inhibition of H3K18 acetylation. These findings indicate that autoacetylation of p300 greatly reduces its ability to acetylate H3K18. The results reported here combined with our previous findings suggest that E1A can repress transcription by multiple strategies, including altering the chromatin modifying activity of p300 and dissociating TFIID from the TATA box thus disrupting formation of the transcription pre-initiation complex [5, 6].

No MeSH data available.


Related in: MedlinePlus

Enhancement of p300 autoacetylation by E1A 1-80 and correlation with inhibition of H3K18 acetylationA. p300 autoacetylation is enhanced by E1A 1-80. In vitro acetylation assay was performed with p300, VP16 (Gal4-VP16) (lanes 3 and 5), E1A 1-80 (264 nM, lanes 4 and 5), or C6A mutant (lane 6) in the absence of chromatin. Western blots were performed with acetyl-Lys antibody (for acetylated p300, Ac-p300, upper panel) or p300 antibody (lower panel).B. Quantitation of autoacetylated p300. Western blot chemiluminescence was quantitated on a Bio-Rad scanner directly (see “Materials and methods”), and normalized to the Ac-p300 level of lane 2. Data from lanes 2-6 were plotted.C. Titration of E1A 1-80 for its modulation of p300 autoacetylation and H3K18 acetylation. In vitro acetylation reaction was performed with p300 and VP16 (Gal4-VP16) with assembled chromatin, and varying amounts of E1A 1-80 (lanes 3-6), or C6A mutant (lane 7). Western blots were performed with antibodies as indicated.D. Quantitation of p300 autoacetylation and H3K18 acetylation. Quantitation of Western blot signals was as in B. Data from lanes 2-5 were plotted.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4362482&req=5

Figure 2: Enhancement of p300 autoacetylation by E1A 1-80 and correlation with inhibition of H3K18 acetylationA. p300 autoacetylation is enhanced by E1A 1-80. In vitro acetylation assay was performed with p300, VP16 (Gal4-VP16) (lanes 3 and 5), E1A 1-80 (264 nM, lanes 4 and 5), or C6A mutant (lane 6) in the absence of chromatin. Western blots were performed with acetyl-Lys antibody (for acetylated p300, Ac-p300, upper panel) or p300 antibody (lower panel).B. Quantitation of autoacetylated p300. Western blot chemiluminescence was quantitated on a Bio-Rad scanner directly (see “Materials and methods”), and normalized to the Ac-p300 level of lane 2. Data from lanes 2-6 were plotted.C. Titration of E1A 1-80 for its modulation of p300 autoacetylation and H3K18 acetylation. In vitro acetylation reaction was performed with p300 and VP16 (Gal4-VP16) with assembled chromatin, and varying amounts of E1A 1-80 (lanes 3-6), or C6A mutant (lane 7). Western blots were performed with antibodies as indicated.D. Quantitation of p300 autoacetylation and H3K18 acetylation. Quantitation of Western blot signals was as in B. Data from lanes 2-5 were plotted.

Mentions: In vitro acetylation reactions were performed with p300, Gal4-VP16, E1A 1-80 and its C6A mutant in different combinations in the absence of chromatin (Figure 2A). Reaction products were examined for acetylated p300 (Ac-p300) by Western blot analysis with an antibody against acetylated lysine. E1A 1-80 significantly enhanced p300 autoacetylation in the absence of Gal4-VP16 (Figure 2A, upper panel, lane 4,) or in the presence of Gal4-VP16 (upper panel, lane 5) (see Figure 2B for quantitation). In contrast, the C6A mutant was substantially defective in enhancement of p300 autoacetylation (upper panel, lane 6). p300 levels were constant among the different assays as measured by Western blot analysis (Figure 2A, lower panel).


The adenovirus E1A oncoprotein N-terminal transcriptional repression domain enhances p300 autoacetylation and inhibits histone H3 Lys18 acetylation.

Zhao LJ, Loewenstein PM, Green M - Genes Cancer (2015)

Enhancement of p300 autoacetylation by E1A 1-80 and correlation with inhibition of H3K18 acetylationA. p300 autoacetylation is enhanced by E1A 1-80. In vitro acetylation assay was performed with p300, VP16 (Gal4-VP16) (lanes 3 and 5), E1A 1-80 (264 nM, lanes 4 and 5), or C6A mutant (lane 6) in the absence of chromatin. Western blots were performed with acetyl-Lys antibody (for acetylated p300, Ac-p300, upper panel) or p300 antibody (lower panel).B. Quantitation of autoacetylated p300. Western blot chemiluminescence was quantitated on a Bio-Rad scanner directly (see “Materials and methods”), and normalized to the Ac-p300 level of lane 2. Data from lanes 2-6 were plotted.C. Titration of E1A 1-80 for its modulation of p300 autoacetylation and H3K18 acetylation. In vitro acetylation reaction was performed with p300 and VP16 (Gal4-VP16) with assembled chromatin, and varying amounts of E1A 1-80 (lanes 3-6), or C6A mutant (lane 7). Western blots were performed with antibodies as indicated.D. Quantitation of p300 autoacetylation and H3K18 acetylation. Quantitation of Western blot signals was as in B. Data from lanes 2-5 were plotted.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362482&req=5

Figure 2: Enhancement of p300 autoacetylation by E1A 1-80 and correlation with inhibition of H3K18 acetylationA. p300 autoacetylation is enhanced by E1A 1-80. In vitro acetylation assay was performed with p300, VP16 (Gal4-VP16) (lanes 3 and 5), E1A 1-80 (264 nM, lanes 4 and 5), or C6A mutant (lane 6) in the absence of chromatin. Western blots were performed with acetyl-Lys antibody (for acetylated p300, Ac-p300, upper panel) or p300 antibody (lower panel).B. Quantitation of autoacetylated p300. Western blot chemiluminescence was quantitated on a Bio-Rad scanner directly (see “Materials and methods”), and normalized to the Ac-p300 level of lane 2. Data from lanes 2-6 were plotted.C. Titration of E1A 1-80 for its modulation of p300 autoacetylation and H3K18 acetylation. In vitro acetylation reaction was performed with p300 and VP16 (Gal4-VP16) with assembled chromatin, and varying amounts of E1A 1-80 (lanes 3-6), or C6A mutant (lane 7). Western blots were performed with antibodies as indicated.D. Quantitation of p300 autoacetylation and H3K18 acetylation. Quantitation of Western blot signals was as in B. Data from lanes 2-5 were plotted.
Mentions: In vitro acetylation reactions were performed with p300, Gal4-VP16, E1A 1-80 and its C6A mutant in different combinations in the absence of chromatin (Figure 2A). Reaction products were examined for acetylated p300 (Ac-p300) by Western blot analysis with an antibody against acetylated lysine. E1A 1-80 significantly enhanced p300 autoacetylation in the absence of Gal4-VP16 (Figure 2A, upper panel, lane 4,) or in the presence of Gal4-VP16 (upper panel, lane 5) (see Figure 2B for quantitation). In contrast, the C6A mutant was substantially defective in enhancement of p300 autoacetylation (upper panel, lane 6). p300 levels were constant among the different assays as measured by Western blot analysis (Figure 2A, lower panel).

Bottom Line: Additional acetylation of p300 in the presence of E1A 1-80 produces stronger inhibition of H3K18 acetylation.These findings indicate that autoacetylation of p300 greatly reduces its ability to acetylate H3K18.The results reported here combined with our previous findings suggest that E1A can repress transcription by multiple strategies, including altering the chromatin modifying activity of p300 and dissociating TFIID from the TATA box thus disrupting formation of the transcription pre-initiation complex [5, 6].

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Virology, Saint Louis University School of Medicine, Doisy research Center, St. Louis, Missouri.

ABSTRACT
Expression of the adenovirus E1A N-terminal transcription repression domain alone (E1A 1-80) represses transcription from specific promoters such as HER2 [1] and from reconstituted chromatin [2]. Significantly, E1A 1-80 can induce the death of human breast cancer cells over-expressing the HER2 oncogene [1] as well as other cancer cells. Here, we report that E1A 1-80 alone is sufficient to inhibit H3K18 acetylation in vivo and p300-mediated H3K18 acetylation in reconstituted chromatin. Of interest, hypoacetylation of H3K18 has been correlated with the survival of tumor cells and the poor prognosis of many cancers [3, 4]. E1A 1-80 enhances p300 autoacetylation and concurrently inhibits H3K18 acetylation in chromatin in a dose-dependent manner. Pre-acetylation of p300 by incubation with acetyl-CoA alone reduces p300's ability to acetylate H3K18 in chromatin. Additional acetylation of p300 in the presence of E1A 1-80 produces stronger inhibition of H3K18 acetylation. These findings indicate that autoacetylation of p300 greatly reduces its ability to acetylate H3K18. The results reported here combined with our previous findings suggest that E1A can repress transcription by multiple strategies, including altering the chromatin modifying activity of p300 and dissociating TFIID from the TATA box thus disrupting formation of the transcription pre-initiation complex [5, 6].

No MeSH data available.


Related in: MedlinePlus