LXCXE-independent chromatin remodeling by Rb/E2f mediates neuronal quiescence

Abstract

Neuronal survival is dependent upon the retinoblastoma family members, Rb1 (Rb) and Rb2 (p130). Rb is thought to regulate gene repression, in part, through direct recruitment of chromatin modifying enzymes to its conserved LXCXE binding domain. We sought to examine the mechanisms that Rb employs to mediate cell cycle gene repression in terminally differentiated cortical neurons. Here, we report that Rb loss converts chromatin at the promoters of E2f-target genes to an activated state. We established a mouse model system in which Rb-LXCXE interactions could be induciblely disabled. Surprisingly, this had no effect on survival or gene silencing in neuronal quiescence. Absence of the Rb LXCXE-binding domain in neurons is compatible with gene repression and long-term survival, unlike Rb deficiency. Finally, we are able to show that chromatin activation following Rb deletion occurs at the level of E2fs. Blocking E2f-mediated transcription downstream of Rb loss is sufficient to maintain chromatin in an inactive state. Taken together our results suggest a model whereby Rb-E2f interactions are sufficient to maintain gene repression irrespective of LXCXE-dependent chromatin remodeling.

Keywords: E2f; Rb; cell cycle; neuronal quiescence; transcription.

Figure 1. Acute Rb removal results chromatin activation at E2f-regulated promoters. qRT-PCR analysis of the PCNA and CycA2 promoters in GFP- or Cre-infected cortical neurons (Rb^Flox/Flox) at 6 DIV from chromatin immunoprecipitated DNA. (A) Western blot analysis of total protein extracted from control (GFP) and Rb-deficient (Cre) cortical neurons at 6DIV. (B) Chromatin was immunoprecipitated with an antibody directed toward acetylated histone 3 (Acetyl-H3). (C) Chromatin was immunoprecipitated using an antibody that recognizes trimethylated lysine 9 on histone 3 (H3K9me3). Error bars represent SEM (n = 3, where n represents a ChIP performed on neurons isolated from a unique embryo), significance was determined using a two-tailed Student’s t-test where *p < 0.05.

Figure 2. Rb regulates neuronal quiescence in an LXCXE-independent manner. (A) Western blot analysis of total protein extracted from control, Rb-deficient and Rb^ΔL cortical neurons at 10 DIV (B) Cortical neurons were fixed at 10DIV and stained for Ki67. Percentage of Ki67+ was quantified using DAPI to represent total cell number. Error bars represent SEM (n = 3, where n represent an independent culture with multiple technical replicates); statistical significance was determined using a one-way ANOVA, where *p < 0.05 was considered statistically significant. (C) Animals of indicated genotypes were injected with tamoxifen (180 mg/kg/day i.p., 3 d) and euthanized 4 wk following the final injection. Representative pictures of ectopic expression of the cell cycle-related factors Ki67 (arrows) and Cyclin E (arrows) in neurons in the cortex of CamKCreERT2; Rb^Flox/Floxmice compared with CamKCreERT2; Rb^Flox/+ and CamKCreERT2; Rb^Flox/ΔLmice. Error bars represent SEM (n = 3, where n represent the cortex from a unique animal with multiple technical replicates); statistical significance was determined using a one-way ANOVA, where *p < 0.05 was considered statistically significant. Scale bar: 50 µM.

Figure 3. Rb regulates neuronal survival in an LXCXE independent manner. (A) Western blot analysis of total protein extracted from control, Rb-deficient and Rb^ΔL cortical neurons at 10 DIV (B) Cortical neurons of the indicated genotypes were infected with lentiviral Cre and fixed at 10 DIV, and condensed nuclei were examined by DAPI staining. Error bars represent SEM (n = 3, where n represent an independent culture with multiple technical replicates); statistical significance was determined using a one-way ANOVA, where *p < 0.05 was considered statistically significant. (C) Animals of indicated genotypes were injected with tamoxifen (180 mg/kg/day i.p., 3 d) and euthanized 4 wk following the final injection. Representative pictures of NeuN immunofluorescence in the cortex of CamKCreERT2; Rb^Flox/Floxmice compared with CamKCreERT2; Rb^Flox/+ and CamKCreERT2; Rb^Flox/ΔLmice. Scale bar: 100 µm. (D) NeuN+ cells were quantified in the cortex, and percentages were obtained by normalizing to total DAPI cells for indicated genotypes. Error bars represent SEM (n = 3, where n represents the cortex from a unique animal with multiple technical replicates); statistical significance was determined using a one-way ANOVA, where *p < 0.05 was considered statistically significant.

Figure 4. Chromatin activation in the absence of Rb occurs at the level of E2f. qRT-PCR analysis of the PCNA and CycA2 promoters in GFP, Cre or Cre/ DP1_Δ103–126 infected cortical neurons (Rb^Flox/Flox) at 6DIV from chromatin immunoprecipitated DNA. (A) Western blot analysis of total protein extracted from control (GFP), Rb-deficient (Cre) and Rb-deficient/dominant negative DP1 (Cre and DP1_Δ103–126) cortical neurons at 6 DIV. (B) Chromatin was immunoprecipitated with an antibody directed toward acetylated histone 3 (Acetyl-H3). (C) Chromatin was immunoprecipitated using an antibody that recognizes tri-methylated lysine 9 on histone 3 (H3K9me3). Error bars represent SEM (n = 3, where n represents a ChIP performed on neurons isolated from a unique embryo); statistical significance was determined using a one-way ANOVA, where *p < 0.05 was considered statistically significant.