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ATF2 is required for amino acid-regulated transcription by orchestrating specific histone acetylation.

Bruhat A, Chérasse Y, Maurin AC, Breitwieser W, Parry L, Deval C, Jones N, Jousse C, Fafournoux P - Nucleic Acids Res. (2007)

Bottom Line: Using ATF2-deficient mouse embryonic fibroblasts, we demonstrate that ATF2 is essential in the acetylation of histone H4 and H2B in vivo.The role of ATF2 on histone H4 acetylation is dependent on its binding to the AARE and can be extended to other amino acid regulated genes.Thus, ATF2 is involved in promoting the modification of the chromatin structure to enhance the transcription of a number of amino acid-regulated genes.

View Article: PubMed Central - PubMed

Affiliation: UMR 1019, Unité de Nutrition Humaine, INRA de Theix, 63122 Saint Genès Champanelle, France. bruhat@clermont.inra.fr

ABSTRACT
The transcriptional activation of CHOP (a CCAAT/enhancer-binding protein-related gene) by amino acid deprivation involves the activating transcription factor 2 (ATF2) and the activating transcription factor 4 (ATF4) binding the amino acid response element (AARE) within the promoter. Using a chromatin immunoprecipitation approach, we report that in vivo binding of phospho-ATF2 and ATF4 to CHOP AARE are associated with acetylation of histones H4 and H2B in response to amino acid starvation. A time course analysis reveals that ATF2 phosphorylation precedes histone acetylation, ATF4 binding and the increase in CHOP mRNA. We also show that ATF4 binding and histone acetylation are two independent events that are required for the CHOP induction upon amino acid starvation. Using ATF2-deficient mouse embryonic fibroblasts, we demonstrate that ATF2 is essential in the acetylation of histone H4 and H2B in vivo. The role of ATF2 on histone H4 acetylation is dependent on its binding to the AARE and can be extended to other amino acid regulated genes. Thus, ATF2 is involved in promoting the modification of the chromatin structure to enhance the transcription of a number of amino acid-regulated genes.

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Role of ATF2 in histone acetylation in response to leucine deprivation. (A) ATF2 +/+ and ATF2 −/− MEF 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 using antibodies specific for ATF4, ATF2, phospho-ATF2 (Thr-71), acetylated H3, acetylated H4 and acetylated H2B and a set of primers to produce amplicon B (Figure 2A). 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 standard error of the means. (B) The same experiment as described in (A) was also performed with wild ATF4 +/+ and ATF4 −/− MEF.
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Figure 4: Role of ATF2 in histone acetylation in response to leucine deprivation. (A) ATF2 +/+ and ATF2 −/− MEF 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 using antibodies specific for ATF4, ATF2, phospho-ATF2 (Thr-71), acetylated H3, acetylated H4 and acetylated H2B and a set of primers to produce amplicon B (Figure 2A). 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 standard error of the means. (B) The same experiment as described in (A) was also performed with wild ATF4 +/+ and ATF4 −/− MEF.

Mentions: The results described above suggest that ATF2 could play a critical role in the acetylation of histone and thus in the onset of CHOP transcription upon amino acid starvation. To investigate the link between ATF2 and acetylation of histones, ChIP experiments were performed in MEFs deficient in ATF2 and in the corresponding wild type cells incubated either in control or in leucine-starved medium for 2 h. The ChIP results obtained with wild type MEFs (Figure 4A) are consistent with those described above with HeLa cells (Figure 2B and C). In ATF2-deficient cells, no ATF2 or phosphorylated ATF2 bound to the CHOP AARE is detected. However, the increase in the ATF4 binding remains (Figure 4A). Furthermore, in the absence of ATF2, the increase in histone H4 and H2B acetylation in response to amino acid starvation was lost. The same result was obtained with cells starved for 4 h with leucine (data not shown). ATF2 therefore is essential for the acetylation of histones H4 and H2B and thereby plays a crucial role in the modification of the chromatin structure associated with activation of CHOP transcription. By contrast, in cells lacking ATF4, the levels of ATF2 phosphorylation and of histone H4 and H2B acetylation are unchanged (Figure 4B). Taken together, these results demonstrate that histone acetylation and ATF4 binding are two independent events that are required for the CHOP induction upon amino acid starvation. It is noticeable that it remains an ATF2-independent level of acetylated histone. ChIP experiments were performed from ATF2 KO cells with primer sets covering much farther upstream or downstream from the CHOP gene (data not shown). The results indicated that the amount of histone acetylation in ATF2-deficient cells is due to the background observed for each histone antibody.Figure 4.


ATF2 is required for amino acid-regulated transcription by orchestrating specific histone acetylation.

Bruhat A, Chérasse Y, Maurin AC, Breitwieser W, Parry L, Deval C, Jones N, Jousse C, Fafournoux P - Nucleic Acids Res. (2007)

Role of ATF2 in histone acetylation in response to leucine deprivation. (A) ATF2 +/+ and ATF2 −/− MEF 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 using antibodies specific for ATF4, ATF2, phospho-ATF2 (Thr-71), acetylated H3, acetylated H4 and acetylated H2B and a set of primers to produce amplicon B (Figure 2A). 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 standard error of the means. (B) The same experiment as described in (A) was also performed with wild ATF4 +/+ and ATF4 −/− MEF.
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Figure 4: Role of ATF2 in histone acetylation in response to leucine deprivation. (A) ATF2 +/+ and ATF2 −/− MEF 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 using antibodies specific for ATF4, ATF2, phospho-ATF2 (Thr-71), acetylated H3, acetylated H4 and acetylated H2B and a set of primers to produce amplicon B (Figure 2A). 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 standard error of the means. (B) The same experiment as described in (A) was also performed with wild ATF4 +/+ and ATF4 −/− MEF.
Mentions: The results described above suggest that ATF2 could play a critical role in the acetylation of histone and thus in the onset of CHOP transcription upon amino acid starvation. To investigate the link between ATF2 and acetylation of histones, ChIP experiments were performed in MEFs deficient in ATF2 and in the corresponding wild type cells incubated either in control or in leucine-starved medium for 2 h. The ChIP results obtained with wild type MEFs (Figure 4A) are consistent with those described above with HeLa cells (Figure 2B and C). In ATF2-deficient cells, no ATF2 or phosphorylated ATF2 bound to the CHOP AARE is detected. However, the increase in the ATF4 binding remains (Figure 4A). Furthermore, in the absence of ATF2, the increase in histone H4 and H2B acetylation in response to amino acid starvation was lost. The same result was obtained with cells starved for 4 h with leucine (data not shown). ATF2 therefore is essential for the acetylation of histones H4 and H2B and thereby plays a crucial role in the modification of the chromatin structure associated with activation of CHOP transcription. By contrast, in cells lacking ATF4, the levels of ATF2 phosphorylation and of histone H4 and H2B acetylation are unchanged (Figure 4B). Taken together, these results demonstrate that histone acetylation and ATF4 binding are two independent events that are required for the CHOP induction upon amino acid starvation. It is noticeable that it remains an ATF2-independent level of acetylated histone. ChIP experiments were performed from ATF2 KO cells with primer sets covering much farther upstream or downstream from the CHOP gene (data not shown). The results indicated that the amount of histone acetylation in ATF2-deficient cells is due to the background observed for each histone antibody.Figure 4.

Bottom Line: Using ATF2-deficient mouse embryonic fibroblasts, we demonstrate that ATF2 is essential in the acetylation of histone H4 and H2B in vivo.The role of ATF2 on histone H4 acetylation is dependent on its binding to the AARE and can be extended to other amino acid regulated genes.Thus, ATF2 is involved in promoting the modification of the chromatin structure to enhance the transcription of a number of amino acid-regulated genes.

View Article: PubMed Central - PubMed

Affiliation: UMR 1019, Unité de Nutrition Humaine, INRA de Theix, 63122 Saint Genès Champanelle, France. bruhat@clermont.inra.fr

ABSTRACT
The transcriptional activation of CHOP (a CCAAT/enhancer-binding protein-related gene) by amino acid deprivation involves the activating transcription factor 2 (ATF2) and the activating transcription factor 4 (ATF4) binding the amino acid response element (AARE) within the promoter. Using a chromatin immunoprecipitation approach, we report that in vivo binding of phospho-ATF2 and ATF4 to CHOP AARE are associated with acetylation of histones H4 and H2B in response to amino acid starvation. A time course analysis reveals that ATF2 phosphorylation precedes histone acetylation, ATF4 binding and the increase in CHOP mRNA. We also show that ATF4 binding and histone acetylation are two independent events that are required for the CHOP induction upon amino acid starvation. Using ATF2-deficient mouse embryonic fibroblasts, we demonstrate that ATF2 is essential in the acetylation of histone H4 and H2B in vivo. The role of ATF2 on histone H4 acetylation is dependent on its binding to the AARE and can be extended to other amino acid regulated genes. Thus, ATF2 is involved in promoting the modification of the chromatin structure to enhance the transcription of a number of amino acid-regulated genes.

Show MeSH