Skip to main content
. 2019 Oct 28;294(49):18674–18684. doi: 10.1074/jbc.RA119.010447

Figure 4.

Figure 4.

Promoter hypermethylation restricts ATF4-dependent induction of ASNS following asparagine depletion. A, bisulfite sequencing analyses revealed that the ASNS promoter is hypermethylated in RS4;11 cells. As a control, the same region is hypomethylated in Nalm-6 cells. Each dot represents a predicted CpG site. Each line represents one sequencing result from a bulk population of cells. B, RS4;11 cells were maintained in medium containing low levels of exogenous asparagine (20 μm, 20% of complete LCM) for 4 weeks. The resulting cells were then plated on 1% methylcellulose prepared in asparagine-free LCM for another 4 weeks to establish the RS4;11/R line, which was maintained in asparagine-deficient LCM. C, growth curve of RS4;11 and RS4;11/R cells cultured in in asparagine-replete or -depleted medium for 4 days. Results are presented as mean ± S.D. of triplicates from a representative experiment. D, RS4;11 and RS4;11/R cells were cultured in asparagine-replete or -depleted medium for 24 h. Protein lysates were analyzed by immunoblot with antibodies specific for p-GCN2 (Thr-899), GCN2, ATF4, and ASNS. E, RS4;11 and RS4;11/R cells were cultured under the same condition as in D. RNA was extracted and subjected to qPCR analysis. The result is normalized to 18S rRNA as an internal control. Results are presented as mean ± S.D. of triplicates from a representative experiment. F, bisulfite sequencing results of the ASNS promoter from a bulk population of RS4;11/R cells. G, RS4;11 and RS4;11/R cells were cultured in asparagine-replete or -depleted medium for 24 h. A ChIP assay was performed using an ATF4-specific antibody or an isotype IgG control antibody. Promoter-specific qPCR amplification was done with primer pairs specific for the promoter region of ASNS (left) or SESN2 (right). Results were normalized to total input DNA prior to antibody enrichment. Data are presented as mean ± S.D. of triplicates from a representative experiment. The p values were calculated using Student's two-tailed paired t test. **, p < 0.01; ***, p < 0.001; ns, not significant. H, RS4;11/R cells stably expressing control guide RNA (sgCtrl) or ATF4-specific guide RNA were cultured in asparagine-replete or -depleted medium for 24 h. Protein lysates were analyzed by immunoblot analyses for ATF4 and ASNS.