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. 2016 Jul 18;44(20):9667–9680. doi: 10.1093/nar/gkw643

Figure 3.

Figure 3.

Impact of DNA damage and CREB S111 phosphorylation on coactivator recruitment and DNA binding activity. (A) ChIP analysis of CREB, CBP and CRTC2 over the CREM promoter in CREB+/+ and CREBS111A MEFs. Cells were treated with Fsk for 90 min, irradiated with 20 Gy IR for 120 min (20 Gy) or treated with 20 Gy IR 30 min prior to Fsk (20 Gy-Fsk) and processed for ChIP-qPCR using the indicated antibodies. Y-axis represents the CREM promoter occupancy. Each bar represents averaged results, n = 3. Error bars indicate SEM. *P < 0.05 (Student's t-test). (B) DNA damage inhibits CREB–CRTC2–DNA ternary complex formation in an S111 phosphorylation-dependent manner. HEK 293T cells transfected with CREBWT or CREB3A were treated with 2 nM CLM for 1 h and cell lysates incubated with GST-CRTC2CBD and either CRE-containing or scrambled oligonucleotides in the presence of GSH-agarose beads. Immobilized CREB–CRTC2–DNA ternary complexes were washed under indicated buffer conditions analyzed by western blotting with α-CREB and α-GST antibodies. (C) Immortalized CREB+/+ and CREBS111A MEFs were treated with Fsk for 90 min, treated with 2 nM CLM for 120 min (CLM), or treated with CLM for 30 min prior to Fsk (CLM + Fsk). CREB–CRE complexes were analyzed by electrophoretic mobility shift assay (EMSA). (D) DNA-binding activity was analyzed by EMSA using cell lysates prepared from HEK 293T cells transfected with empty vector, or plasmids encoding CREBWT, CREB3A (100A, 111A and 121A), CREBQ112D or DNA-binding defective KCREB. The transfected cells were irradiated with 50 Gy IR for 1 h and CREB expression analyzed by immunoblotting. (E) CRE binding activity was measured by densitometric analysis. *P < 0.05 (Student's t-test). (F) DNA damage caused dissociation of CREB from bulk chromatin. HeLa cells were irradiated with 10 Gy IR, collected at the indicated times, and fractionated into soluble (Sol) or chromatin (Chr) fractions prior to sodium dodecyl sulphate-polyacrylamide gel electrophoresisand immunoblotting with α-CREB or α-MCM3 antibodies. (G) CREB chromatin dissociation is ATM dependent. HeLa cells were pretreated with DMSO or ATM inhibitor (KU-55933) for 30 min, and then irradiated with 10 Gy IR for 1 h. Soluble (Sol) or chromatin (Chr) fractions were analyzed using α-CREB, α-HSP90, α-MCM3 or α-TDP-43 antibodies. (H) The S111A mutation inhibits IR-dependent CREB chromatin eviction. Chromatin fractionation of CREB+/+ and CREBS111A MEFs was carried out 1 h after irradiation of 10 Gy IR.