Fig 2: Both AD1 and AD2 regions of SRC-3 bind preferentially acetylated Sox2.A, schematic diagrams showing SRC-3 structural and functional domains and three constructs used in this study. B, co-IP assay analyzing the interaction between Sox2 and three distinct regions of SRC-3. HEK293T cells were transfected with FLAG-Sox2 and treated with DMSO or TSA (1 µM) plus NAM (5 mM) (T + N) for 24 h. In addition, HEK293T cells were transfected with Myc-SRC-3-RID, Myc-SRC-3-AD1, or Myc-SRC-3-AD2, respectively. The cellular extracts were prepared and mixed as indicated for IP with anti-Myc and WB analysis using Myc and FLAG antibodies as indicated. Note both SRC-3-AD1 and SRC-3-AD2 regions bind with enhanced affinity for acetylated Sox2. C, co-IP assay comparing the binding of acetylated Sox2 to full-length SRC-3 and SRC-3-AD1 and SRC-3-AD2. The experiments were carried out as in (B). AD1, activation domain 1; AD2, activation domain 2; co-IP, co-immunoprecipitation; DMSO, dimethyl sulfoxide; IP, immunoprecipitation; NAM, nicotinamide; RID, receptor-interaction domain; Sox2, SRY-box 2; SRCs, steroid receptor coactivators; TSA, trichostatin A.

Fig 3: SRC family coactivators enhance Sox2 transcriptional activity.A–C, luciferase reporter assay showing that the SRC family coactivators SRC-1 (A), SRC-2 (B), and SRC-3 (C) enhanced transcriptional activation by Sox2 in a dose-dependent manner. Also shown are WB results of ectopically expressed FLAG-Sox2 with Myc-SRC-1, Myc-SRC-2, or Myc-SRC-3, respectively. GAPDH serves as a loading control. FGF4-Luc reporter, 50 ng; FLAG-Sox2, 200 ng; Myc-SRC-1/2/3, 200 ng, 500 ng, and 1000 ng for an increasing dose of SRC expression plasmids. D–F, luciferase reporter assay showing that SRC-1 (D), SRC-2 (E), and SRC-3 (F) promoted Sox2 transcriptional activity synergistically with p300. Also shown are WB results of ectopically expressed FLAG-Sox2, Myc-p300, and Myc-SRC-1, Myc-SRC-2, or Myc-SRC-3, respectively. FGF4-Luc, 50 ng; FLAG-Sox2, 200 ng; Myc-SRC-1/2/3, 200 ng and 500 ng; Myc-p300, 1000 ng. Data are represented as relative luciferase activity (×104) (mean ± SD of three experimental repeats). All statistical analyses were performed using Excel 2016. *p< 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Sox2, SRY-box 2; SRCs, steroid receptor coactivators.

Fig 4: Sox2 acetylation promotes the interaction between Sox2 and SRC-2/3.A, IP-WB analysis showing that ectopically expressed p300 elevated Sox2 acetylation. B, co-IP assay showing that coexpressed p300 enhanced the interaction between SRC-3 and Sox2. A nonspecific band that overlaps with Myc-Sox2 (*). C, IP-WB analysis showing that inhibition of both histone de-acetylase and Sirtuin by TSA and NAM elevated Sox2 acetylation. FLAG-Sox2 was transiently expressed in HEK239T cells, and cells were treated with DMSO or TSA (1 µM) plus NAM (5 mM) for 24 h before being harvested for IP-WB analysis. D, co-IP assay showing that TSA plus NAM treatment enhanced the interaction between Sox2 and SRC-3. FLAG-SRC-3 and Myc-Sox2 were coexpressed in HEK239T cells, and cells were treated with TSA (1 µM) plus NAM (5 mM) or sodium crotonate (NaCro, 20 mM) for 24 h before being harvested for co-IP assay using the antibody as indicated. E, co-IP assay showing that TSA plus NAM treatment markedly enhanced the interaction between ectopically expressed FLAG-Sox2 and endogenous SRC-3. F, the right panel showing the experimental scheme. FLAG-Sox2–transfected HEK239T cells were treated with DMSO or TSA (1 µM) plus NAM (5 mM) (T + N) for 24 h before being harvested for preparation of whole-cell extracts, and the resulting extracts were mixed with an equal volume of extracts derived from Myc-SRC-3–expressed HEK239T cells for the co-IP assay using the antibodies as indicated. G, the right panel showing the experimental scheme. Myc-SRC-3–transfected HEK239T cells were treated with DMSO or TSA (1 µM) plus NAM (5 mM) (T + N) for 24 h before being harvested for preparation of whole-cell extracts, and the resulting extracts were mixed with an equal volume of extracts derived from FLAG-Sox2–expressed HEK239T cells, followed by the co-IP assay using antibodies as indicated. H, experiments were performed essentially as (F), except that the Myc-Sox2 expression construct was used and that FLAG-SRC-1– or FLAG-SRC-2–expressed HEK239T cell extracts were used for the co-IP assay using antibodies as indicated. I, co-IP assay showing that TSA (1 µM) plus NAM (5 mM) treatment (12 h) markedly enhanced the interaction between endogenous Sox2 and endogenous SRC-2 and SRC-3 in E14 ES cells. J, IP-WB analysis showing that TSA (1 µM) plus NAM (5 mM) treatment elevated the level of Sox2 acetylation in E14 ES cells. ImageJ was used for quantitation of WBs. co-IP, co-immunoprecipitation; IP, immunoprecipitation; NAM, nicotinamide; Sox2, SRY-box 2; SRCs, steroid receptor coactivators; TSA, trichostatin A.

Fig 5: K97, K105, and K111 of Sox2 mediate acetylation-enhanced interaction with SRC-3.A, top panel is a schematic diagram showing all mouse Sox2 acetylation sites reported in reference (35). The lower panel illustrates the structural organization of full-length and truncated Sox2 mutants used in this study. B, co-IP assay mapping the Sox2 region(s) required for acetylation-enhanced interaction with SRC-3. HEK293T cells were transfected with FLAG-tagged Sox2 or truncated mutants and treated with DMSO or TSA (1 µM) plus NAM (5 mM) (T + N) for 24 h before being harvested for preparation of whole cell-extracts, and the resulting extracts were mixed with equal volume of extracts derived from Myc-SRC-3–expressed HEK239T cells for the co-IP assay using antibodies as indicated. Note that only Sox2 1 to 243aa, but not 112 to 243aa and 152 to 319aa, showed enhanced interaction with Myc-SRC-3 upon T + N treatment. Full-length and truncated Sox2 mutants bands (*) and light chain of the antibody (?). C, co-IP assay determining the Sox2 lysine residues required for acetylation-enhanced interaction with SRC-3. The experiments were essentially as in (B) except a panel of Sox2 K to R acetylation site mutants was used. D, luciferase reporter assay showing that Sox2-K97R, Sox2-K105R, and Sox2-K111R mutants exhibited reduced transcriptional activities. Data are represented as relative luciferase activity (×104) (mean ± SD of three biological repeats). E, ChIP assay evaluating the binding of FLAG-Sox2, Sox2-K97R, Sox2-K105R, and Sox2-K111R mutants to the transfected FGF4-luciferase reporter. F, ChIP assay showing the recruitment of endogenous SRC-3 to the transfected FGF4-luciferase reporter by FLAG-Sox2, Sox2-K97R, Sox2-K105R, and Sox2-K111R mutants. All statistical analyses were performed using Excel 2016. *p< 0.05; **p < 0.01; ***p < 0.001. co-IP, co-immunoprecipitation; DMSO, dimethyl sulfoxide; NAM, nicotinamide; n.s., no statistical significance; Sox2, SRY-box 2; SRCs, steroid receptor coactivators; TSA, trichostatin A.