Interplay of bromodomain and histone acetylation in the regulation of p300-dependent genes

Abstract

The bromodomain is an evolutionarily conserved motif found in many transcriptional activators including p300 which contains an intrinsic histone acetyltransferase activity and is a general coactivator for many transcription factors. One mode of bromodomain action is to serve as a binding module to recognize specific acetyl-lysine residue of histones during chromatin remodeling and transcriptional activation. The function of p300 is required for diverse sets of gene expression. However, it is not known whether the p300 bromodomain is involved in the expression of all or only subset of p300-dependent genes. In this study, we examined the impact of either wild type or a bromo-deficient p300 on the expression of several p300-dependant genes. The effects of histone acetylation on the expression of these genes were also assessed by targeting histone deacetylase activities with an inhibitor approach. We show that the impact of these inhibitors on the transcriptional activation of p300-dependent genes are impaired in cells containing the bromo-deficient p300, indicating that the interplay of p300 and histone acetylation in p300-dependent gene transcription requires the bromodomain. We also observed an increase in the expression of bromo-deficient p300 at the level of transcription possibly to compensate for the loss of p300 function. However, the high level of bromo-deficient p300 is not able to maintain the basal level of histone acetylation. Thus, the bromodomain is important for p300 to maintain the basal level of histone acetylation and to induce the transcriptional activation of p300-dependent genes. Nevertheless, the requirement of bromodomain and histone acetylation in p300-dependent gene transcription is determined by a gene specific manner.

Figure 1

Characterization of the wild-type and bromo-deficient p300 in HeLa and SiHa cells. (A) Equal amounts of whole cell extracts (50 µg) from the HeLa or SiHa cells were subjected to western analysis for the wild-type and bromo-deficient p300. The blots were then stripped and reprobed for β-actin as a loading control. (B) Quantification of the western blots was plotted as fold difference in reference to the wild-type p300 in the HeLa cells. Error bars represent the standard deviation of three independent experiments. Statistical significance is denoted by * to indicate p < 0.05 compared with the wild-type p300. (C) Relative mRNA level of bromo-deficient p300 in the SiHa cells was analyzed by quantitative real-time RT-PCR and presented as fold variation to the wild-type p300 transcripts in the HeLa cells. Error bars represent standard deviations of three independent experiments in duplicate samples. (D) Relative synthesis of the bromo-deficient p300 protein in the SiHa cells was determined following 2 h of pulse-labeling with ³⁵S-methionine and expressed as fold variation of the wild-type p300 in the HeLa cells. Error bars represent standard deviations of three independent experiments. (E) The HeLa cells were pulse labeled with ³⁵S-methionine for 2 h and chased for 6–24 h. The apparent half-life of the wild-type p300 protein was derived from three independent experiments in duplicate samples. (F) The experimental procedure was as in (E) except that the SiHa cells were used to determine the apparent half-life of the bromo-deficient p300. (G) Intracellular localization of the wild-type or bromodeficient p300 protein in the HeLa or SiHa cells was determined by immunofluorescence microscopy.

Figure 2

Effects of HDAC inhibitors on the expression of p300-dependent genes. (A) HeLa cells were treated with butyrate (NaB, 5 mM), valproic acid (VPA, 5 mM) or TSA (200 nM) for periods of 4, 8 or 16 h. Equal amounts of whole cell extracts (50 µg) were subjected to western analysis of p21, Egr1 or E2F1 protein. The blots were stripped and reprobed for β-actin as loading controls for quantification. (B) Quantification of the western blots was plotted as fold difference in reference to the untreated controls. (C and D) SiHa cells were treated and subjected to western analysis as for the HeLa cells in (A and B).

Figure 3

Effects of HDAC inhibitors on the transcription of p300-dependent genes. (A) Total RNA was extracted from the HeLa cells following 4 or 16 h treatments with butyrate (NaB, 5 mM), valproic acid (VPA, 5 mM) or TSA (200 nM) and reverse transcribed. The relative mRNA levels of p21 or Egr1 were determined by quantitative real time RT-PCR. 18S rRNA was used as an internal control. Values correspond to fold difference in reference to the untreated controls. Error bars present standard deviations of three independent experiments in duplicates. (B) The experimental procedure was as in (A) except that the SiHa cells were used for the real time RT-PCR analysis.

Figure 4

Global histone acetylation and p300-dependent gene expression. (A) Western analysis was performed by using specific antibody against acetylated histone 3 at K9/14 residue following 4 h of treatments of the HeLa or SiHa cells with butyrate (NaB, 5 mM), valproic acid (VPA, 5 mM) or TSA (200 nM). The blots were stripped and reprobed for β-actin as a loading control. (B) Quantification of the western blots is plotted as fold difference in reference to the untreated control of the HeLa cells. Error bars represent standard deviations of three independent experiments. Statistical significance is denoted by * to indicate p < 0.05 compared with the untreated control of HeLa cells. (C) The SiHa cells were transfected with expression plasmid (3 µg) for the full length p300, treated with butyrate, valproic acid or TSA for 8 h and then subjected to western analysis of p21 and Egr1. The blots were stripped and reprobed for β-tubulin as a loading control.