ATF4-dependent regulation of the JMJD3 gene during amino acid deprivation can be rescued in Atf4-deficient cells by inhibition of deacetylation

Abstract

Following amino acid deprivation, the amino acid response (AAR) induces transcription from specific genes through a collection of signaling mechanisms, including the GCN2-eIF2-ATF4 pathway. The present report documents that the histone demethylase JMJD3 is an activating transcription factor 4 (ATF4)-dependent target gene. The JMJD3 gene contains two AAR-induced promoter activities and chromatin immunoprecipitation (ChIP) analysis showed that the AAR leads to enhanced ATF4 recruitment to the C/EBP-ATF response element (CARE) upstream of Promoter-1. AAR-induced histone modifications across the JMJD3 gene locus occur upon ATF4 binding. Jmjd3 transcription is not induced in Atf4-knock-out cells, but the AAR-dependent activation was rescued by inhibition of histone deacetylation with trichostatin A (TSA). The TSA rescue of AAR activation in the absence of Atf4 also occurred for the Atf3 and C/EBP homology protein (Chop) genes, but not for the asparagine synthetase gene. ChIP analysis of the Jmjd3, Atf3, and Chop genes in Atf4 knock-out cells documented that activation of the AAR in the presence of TSA led to specific changes in acetylation of histone H4. The results suggest that a primary function of ATF4 is to recruit histone acetyltransferase activity to a sub-set of AAR target genes. Thus, absolute binding of ATF4 to these particular genes is not required and no ATF4 interaction with the general transcription machinery is necessary. The data are consistent with the hypothesis that ATF4 functions as a pioneer factor to alter chromatin structure and thus, enhance transcription in a gene-specific manner.

FIGURE 1.

AAR-induced expression of JMJD3 mRNA in cells and tissue. Panel A, JMJD3 mRNA content was measured in human hepatoma cell lines (HepG2 or SNU475), primary cultures of freshly isolated human hepatocytes, or in a human non-transformed hepatocyte cell line (HC-04). The AAR was triggered by incubating the cells in DMEM ± HisOH for 8 h prior to isolation of RNA and analysis by qPCR. The data are represented as the averages ± S.D. for at least three samples and are expressed relative to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal control. Panel B, human lung fibroblasts in culture were incubated in DMEM ± HisOH for 8 h prior to analysis of JMJD3 and GAPDH mRNA by qPCR. The data are represented as the averages ± S.D. for at least three samples. The asterisks denote a significant difference of p ≤ 0.05 relative to the DMEM control. Panel C, as described in “Materials and Methods,” mice were either injected I.V. with asparaginase (ASNase) (left panel) or fed a low protein diet (right panel) to induce the AAR in vivo. Liver RNA was analyzed for Jmjd3 and Gapdh mRNA by qPCR. For the ASNase study, the data are the averages ± S.D. for at least three animals. For the low protein diet study, the bars represent the average of two animals and the actual value of each animal is shown as block dots. For all panels, the asterisks denote a significant difference of p ≤ 0.05 relative to the control.

FIGURE 2.

Increased transcription contributes to the induction of JMJD3 expression. Panel A, in HepG2 hepatoma cells, the time course of transcription activity and steady state mRNA content for JMJD3 was measured by qPCR, as described in “Materials and Methods.” The cells were incubated in DMEM ± HisOH for the time indicated and the data are represented as the averages ± S.D. for at least three samples. Panel B, HepG2 protein content for JMJD3 was measured by immunoblotting nuclear extract samples collected 24 h after activating the AAR as described for panel A. To illustrate equal loading, the appropriate section of the blot stained with Fast Green is shown.

FIGURE 3.

The enrichment of binding for transcription factors at the JMJD3 gene as detected by ChIP. Panel A, drawing depicts the gene structure of JMJD3 and the location of five primer sets used to analyze specific gene regions (R1-R5). Primer sequences are listed in supplemental Table 1. Exon numbers are designated within the boxes. Panel B, HepG2 cells were incubated in DMEM ± HisOH for 6 h prior to ChIP analysis for the enrichment of RNA Pol II, ATF4, ATF3, and C/EBPβ. A nonspecific IgG was used as one negative control and the binding at Exon 10 (primer set R5) was taken as “background” for each antibody. The data are represented as the averages ± S.D. for at least three samples, and the asterisks denote a significant difference of p ≤ 0.05 relative to the DMEM control.

FIGURE 4.

Induction of JMJD3 mRNA requires MEK-ERK signaling. Panel A, HEK293T cells were transfected with a control vector (Con) or a vector encoding a constitutively active MEK (+MEK) construct. After 36 h post-transfection, the cells were incubated in DMEM ± HisOH for 8 h prior to analysis of JMJD3 and GAPDH mRNA by qPCR. The asterisks denote a significant difference of p ≤ 0.05. Panel B, HepG2 cells ere incubated for 8 h with or without HisOH in the absence (Control) or presence (MEK inhibitor) of 50 μm PD98059. Panel C, for the HEK293 cells described in panel A, analysis of total and p-ERK by immunoblotting was used as a control to demonstrate MEK signaling after transfection.

FIGURE 5.

The AAR-induced change in histone modifications across the JMJD3 gene locus. The five primer sets (R1-R5), shown in Fig. 3A, were used to scan the JMJD3 gene for changes in histone modifications during the AAR. HepG2 cells were incubated in DMEM ± HisOH for 6 h prior to ChIP analysis for the indicated histone form. The data are represented as the averages ± S.D. for at least three samples. The asterisks denote a significant difference of p ≤ 0.05 relative to the DMEM control.

FIGURE 6.

For selected genes, loss of AAR-induced expression by Atf4 deficiency is rescued by TSA treatment. Wild type (WT) and Atf4-deficient (Atf4-KO) mouse embryonic fibroblasts were incubated in medium containing the HDAC inhibitor TSA for 24 h. During the last 8 h, the cells were incubated in DMEM ± HisOH as indicated. Total RNA was isolated and analyzed by qPCR for the mRNA content of the indicated gene relative to the GAPDH internal control. For each gene, all data were normalized to the DMEM control value in the wild type cells and are shown as the averages ± S.D. for at least three samples. The asterisks denote a significant difference of p ≤ 0.05. Panel A illustrates the data fro the TSA-responsive genes, whereas panel B shows the results for two genes that were not rescued by TSA treatment.

FIGURE 7.

TSA treatment of Atf4-deficient cells results in an increase in H4 acetylation at specific regions within the AAR target genes. Panel A, drawing depicts the gene structure of JMJD3 and the location of seven primer sets used to analyze specific gene regions (R1-R7). Primer sequences are listed in supplemental Table S1. Exon numbers (E) are designated within the boxes. Panel B, MEF cells lacking Atf4 were treated with DMSO or TSA ± HisOH for 1 h and then analyzed by ChIP for H4 acetylation and total H3 protein association on the Jmjd3 gene. Panels C and D, ChIP samples used to obtain the data shown in panel B were also analyzed for three regions (R1-R3) on the Asns (panel C)and Atf3 (panel D) genes. For both genes, region R1 is located about 0.5 kb upstream of the transcription start site, region R2 includes the CARE (nt −68 for Asns and nt −23 for Atf3) and the proximal promoter, and region R3 is located downstream within the exon (E) indicated. The data are represented as the averages ± S.D. for at least three samples, and the asterisks denote a significant difference of p ≤ 0.05 relative to the DMEM control.