Fig. 2. Generation and characterization of DT mice expressing RbAp48-DN in the forebrain.
(A) (Left) Radioactive in situ hybridization of RbAp48 mRNA on a sagittal brain slice from adult wild-type (WT) mice (3.5 months). (Right) High magnification of left image showing RbAp48 mRNA in the hippocampus (hp). See table S2 for hybridization probe sequence (oligo1). (B) Representative Western blot (different mouse in each lane) and averaged values below. RbAp48 values were normalized to tubulin values from the same mouse, and data from all mice were averaged and expressed as fold difference change in aged mice (15 months) compared to young mice (3.5 months) ± SEM. RbAp48 is reduced in the DG of aged mice compared to young mice (*P = 3.2 × 10−8). n = 24 per age (six mice per age; four independent experiments). (C) (a) Schematic representation of doxycycline (dox)–regulated expression of Flag–RbAp48-DN in the forebrain. In DT mice that carried the tetOpromoter–RbAp48-DN and the CaMKIIαpromoter-tTA transgenes, the heterologous transactivator tTA bound to the tetO promoter and activated RbAp48-DN expression only in the forebrain where the CaMKIIα promoter is active. Maintaining the DT mice on doxycycline-containing food inhibits RbAp48-DN transcription because doxycycline prevents the binding of the tTA to the tetO promoter. (Right) RbAp48-DN mRNA in sagittal brain sections from adult (day P95) DT animals with no doxycycline treatment after day P40 (transgene ON; on/off dox diet) or with doxycycline treatment after day P80 to inhibit the transgene expression (transgene OFF; on/off/on dox diet). (b) Diet protocols for DT mice and control littermates used in all studies. (c) Western blot analysis of RbAp48-DN (anti-Flag) and endogenous RbAp48 in the hippocampus of 95-day-old mice. DT on dox, DT mouse treated with doxycycline (RbAp48-DN OFF); DT, mouse not fed with doxycycline in adulthood (RbAp48-DN ON); Control, tetO–RbAp48-DN single transgenic mouse. RbAp48-DN is detected only in DT. Four independent experiments gave similar results. (D) Confocal hippocampal images from adult (3.5 months) DT mouse and tetO–RbAp48-DN (tetO) single transgenic animal (control). (Insets) High-magnification DG images. (E) Binding of RbAp48 to histone 4 (H4). (a) Representative Western blots showing RbAp48 level in adult (3.5 months) hippocampal lysates before (input; left) and after (right) coimmunoprecipitation (IP) with anti-H4 antibody. Immunoprecipitated H4 is also shown (right). DT, RbAp48-DN–expressing mouse; ctl, control littermates. (b) Representative Western blots of immunoprecipitated H4 and coimmunoprecipitated RbAp48 from adult (3.5 months) hippocampal lysates from DT mice kept on doxycycline-containing food (RbAp48-DN OFF) and control littermates kept on or off dox in adulthood. IgG and beads: controls for the specificity of the anti-H4 immunoprecipitations. (Graphs) Averaged data (mean ± SEM). (a) DT: n = 4, ctl: n = 6 (tetO = 2, tTA = 2, WT = 2); (b) DT on dox: n = 3; ctl on dox: n = 3 (tetO = 1, tTA = 1, WT = 1). One experiment per mouse. The values of RbAp48 level after the anti-H4 immunoprecipitation were normalized to the H4 values from the same mouse, and data from all mice were averaged and expressed as fold difference change in DT compared to controls in (a) and fold difference change in DT on dox and controls on dox compared to controls off dox in (b). Significant difference was found between DT and controls in (a) [*P = 2 × 10−6, analysis of variance (ANOVA)].