Figure 6. ATF3 binds to p300 and recruits it to SSP genes.

(A) ATF3 was co-transfected with or without FLAG-p300 for coIP assays using the FLAG antibody.

(B) 293T cell lysates were incubated with IgG or the p300 antibody for coIP assays.

(C) HCT116 cells (ATF3-F) expressing 3× FLAG-conjugated endogenous ATF3 protein (see Figure S6 for the editing strategy) or its control cells (WT) were lysed and incubated with the FLAG M2 affinity agarose for coIP assays.

(D) Immobilized GST or GST-ATF3 protein was incubated with purified recombinant p300 protein for GST-pull-down assays. Arrows indicate GST or GST-ATF3 protein.

(E and F) WT or ATF3-KO RPE cells cultured in the serine/glycine-deprived medium for 0, 8, and 24 h were subjected to ChIP assays using the p300 antibody. The PHGDH enhancer (E) and the PSAT1 promoter region (F) were amplified and quantified by qPCR. The data are presented as mean ± SD (n = 3). *p < 0.05; **p < 0.01; ***p < 0.001; Student’s t test.

(G) The CRISPR-Cas9 technology was used to knock out p300 expression (p300KO) from WT and ATF3-KO HCT116 cells. Loss of p300 expression in p300KO and DKO cells was confirmed by western blotting.

(H and I) Indicated HCT116 cells were cultured in normal or the serine/glycine-deprived medium for 24 h and subjected to qRT-PCR to measure the levels of PHGDH and PSAT1 mRNA. Serine-deprivation-caused fold changes in gene expression were presented. The data are presented as mean ± SD (n = 3).

(J) A model shows that ATF3 induced by ATF4 binds to ATF4 and SSP genes to promote SSP gene expression and serine biosynthesis upon serine deprivation.

See also Figure S6.