The p300/CBP-associated factor (PCAF) is a cofactor of ATF4 for amino acid-regulated transcription of CHOP

Abstract

When an essential amino acid is limited, a signaling cascade is triggered that leads to increased translation of the 'master regulator', activating transcription factor 4 (ATF4), and resulting in the induction of specific target genes. Binding of ATF4 to the amino acid response element (AARE) is an essential step in the transcriptional activation of CHOP (a CCAAT/enhancer-binding protein-related gene) by amino acid deprivation. We set out to identify proteins that interact with ATF4 and that play a role in the transcriptional activation of CHOP. Using a tandem affinity purification (TAP) tag approach, we identified p300/CBP-associated factor (PCAF) as a novel interaction partner of ATF4 in leucine-starved cells. We show that the N-terminal region of ATF4 is required for a direct interaction with PCAF and demonstrate that PCAF is involved in the full transcriptional response of CHOP by amino acid starvation. Chromatin immunoprecipitation analysis revealed that PCAF is engaged on the CHOP AARE in response to amino acid starvation and that ATF4 is essential for its recruitment. We also show that PCAF stimulates ATF4-driven transcription via its histone acetyltransferase domain. Thus PCAF acts as a coactivator of ATF4 and is involved in the enhancement of CHOP transcription following amino acid starvation.

Figure 1.

PCAF interacts directly with the N-terminal region of ATF4. (A) Bacterially expressed GST-ATF4 (amino acids 1–351) fusion protein was immobilized on glutathione beads and incubated with nuclear proteins from leucine-starved HeLa cells (2 h). After extensive washing, proteins bound to the beads were eluted in protein sample buffer and analyzed by western blotting with anti-PCAF (top) or anti-ATF4 (bottom) antibodies. The expression of the endogenous (50 kDa) and the recombinant ATF4 proteins (75 kDa) are visualized. Ten percent of the nuclear extracts used in the pull-down experiments were loaded in the Input lane 3. (B) Schematic representation of the ATF4 deletions employed in the experiments reported in (C) and (D). The basic region/leucine zipper (bZip) and the N-terminal (N-ter region) domains are indicated. (C) Nuclear extracts from leucine-starved HeLa cells (2 h) were used in GST pull-down experiments with bacterially expressed full length ATF4 wt (lane 1) or various deletion mutants fused to GST (lanes 2–5). PCAF bound to the GST–ATF4 constructs was detected by western blot with anti-PCAF antibody. Ten percent of the nuclear extracts used in the pull-down experiments were loaded in the Input lane 7. The amount of GST-fused recombinant proteins was monitored by Coomassie blue staining. (D) In vitro pull-down assay of ³⁵S-labeled PCAF against full-length (lane 2), or two ATF4-deletion mutants (lanes 3 and 4) fused to GST. The input (lane 1) was loaded with the amount of ³⁵S-labeled proteins used in the binding reactions. The amount of GST-fused recombinant proteins was monitored by Coomassie blue staining, and radioactive signals of radiolabeled proteins were analyzed using a phosphorimaging device.

Figure 2.

Measurement of PCAF mRNA and protein accumulation in amino acid-starved cells. HeLa cells were incubated either in control (+) or leucine-free medium (−) and harvested for protein extraction and RNA isolation after the indicated incubation times. (A) Total RNA was extracted and real-time RT–PCR was performed as described in Materials and Methods section. The mRNA induction level is defined as the ratio of the relative mRNA level of leucine-starved cells to that of non-starved cells. (B) PCAF, ATF4 and β-Actin protein contents were analyzed by western blots as described in Materials and Methods section.

Figure 3.

PCAF recruitment to CHOP AARE in response to leucine starvation. (A) Scheme of the human CHOP gene indicating the different amplicons produced for the ChIP analysis: A (−1678 to −1478), B (−472 to −301) and C (+1163 to +1372). The AARE is boxed in gray. (B) HeLa cells were incubated 2 h either in control (+leu) or leucine-free medium (−leu) and harvested. ChIP analysis was performed as described under Materials and Methods section using antibodies specific for PCAF and ATF4 and different sets of primers to produce amplicon A, B or C. Data were plotted as the percentage of antibody binding versus the amount of PCR product obtained using a standardized aliquot of input chromatin. Each point represents the mean value of three independent experiments, and the error bars represent the SEM. (C) Time course of PCAF and ATF4 recruitments during leucine starvation. HeLa cells were incubated either in control (+leu) or leucine-free medium (−leu) and harvested for 0–8 h. ChIP analysis was performed using antibodies specific for PCAF and ATF4 and a set of primers to amplify amplicon B (see above). Total RNA was extracted and the CHOP transcriptional activity was determined by real-time RT–PCR analysis of CHOP pre-mRNA as described under Materials and Methods section. The dotted line represents the increase in CHOP pre-mRNA induction level.

Figure 4.

Role of ATF4 in PCAF recruitment to CHOP AARE in response to leucine starvation. ATF4 +/+ and ATF4 −/− MEF were incubated 2 h either in control (+leu) or leucine-free medium (−leu) and harvested. (A) ChIP analysis was performed as described under Materials and Methods section using antibodies specific for PCAF and ATF4 and a set of primers to produce amplicon B (Figure 3A). Data were plotted as the percentage of antibody binding versus the amount of PCR product obtained using a standardized aliquot of input chromatin. Each point represents the mean value of three independent experiments and the error bars represent the SEM. We note that there remains 5% of ATF4 antibody binding in ATF4 KO cells. ChIP experiments were also performed with primer sets reaching much farther upstream or downstream from the CHOP gene (data not shown). The results indicate that the amount of ATF4 binding in ATF4-deficient cells was due to the background observed for the ATF4 antibody. (B) Western blot analysis of ATF4 and PCAF was performed from nuclear extracts.

Figure 5.

Role of PCAF in the stimulation of ATF4 transcriptional activity. HeLa cells were transiently transfected with a luciferase construct containing two copies of the CHOP AARE inserted 5′ to the TK promoter (2X-CHOP-AARE-TK-LUC), the expression vector for ATF4, PCAF or HAT-defective mutant PCAF (PCAFΔHAT) or the empty vector, as indicated. Two days after transfection, cells were harvested for preparation of cell extracts and determination of LUC activity. Results are given as the ‘fold induction’ relative to the cells transfected with empty vector. For all the transfection experiments presented, a plasmid pCMV-βGal was used as an internal control. Relative luciferase activities were determined as described in Materials and Methods section. Each data represents the mean of at least three independent experiments performed in triplicate.

Figure 6.

Effect of PCAF knockdown or PCAF over-expression on the amino acid regulation of CHOP expression. (A) Effect of PCAF knockdown. HeLa cells were transfected with PCAF siRNA or control siRNA. Two days after siRNA transfection, cells were incubated for 2 h in control (C) or in medium lacking leucine (−leu) and then harvested to extract RNA and proteins. Total RNA was extracted and real-time RT–PCR was performed as described in Materials and Methods section. PCAF and ATF4 protein contents were analyzed by western blot. One day after siRNA transfection, cells were transfected with 2X-CHOP-AARE-TK-LUC reporter construct to measure the AARE-dependent transcription. After two days, cells were incubated for 16 h in control (C) or in medium lacking leucine (−leu) and cells were then harvested. LUC activity was measured as described in Materials and Methods section. (B) Effect of PCAF over-expression. HeLa cells were transiently transfected with Flag-PCAF expression construct (PCAF) or with empty vector (vector). Two days after transfection, cells were incubated for 2 h in control (C) or in medium lacking leucine (−leu) and then harvested to extract RNA and proteins. Total RNA was extracted and real-time RT–PCR was performed as described in Materials and Methods section. Each data represents the mean of at least three independent experiments performed in triplicate. Flag-PCAF and β-Actin protein contents were analyzed by western blot.