Control of cognition and adaptive behavior by the GLP/G9a epigenetic suppressor complex - PubMed (original) (raw)

Control of cognition and adaptive behavior by the GLP/G9a epigenetic suppressor complex

Anne Schaefer et al. Neuron. 2009.

Abstract

The genetic basis of cognition and behavioral adaptation to the environment remains poorly understood. Here we demonstrate that the histone methyltransferase complex GLP/G9a controls cognition and adaptive responses in a region-specific fashion in the adult brain. Using conditional mutagenesis in mice, we show that postnatal, neuron-specific deficiency of GLP/G9a leads to derepression of numerous nonneuronal and neuron progenitor genes in adult neurons. This transcriptional alteration is associated with complex behavioral abnormalities, including defects in learning, motivation, and environmental adaptation. The behavioral changes triggered by GLP/G9a deficiency are similar to key symptoms of the human 9q34 mental retardation syndrome that is associated with structural alterations of the GLP/EHMT1 gene. The likely causal role of GLP/G9a in mental retardation in mice and humans suggests a key role for the GLP/G9a-controlled histone H3K9 dimethylation in regulation of brain function through maintenance of the transcriptional homeostasis in adult neurons.

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Figures

Figure 1

Figure 1. Conditional ablation of GLP/G9a in postnatal neurons in the forebrain

(A) Strategy for GLP gene targeting. The partial map of the GLP locus and the map of GLP targeting vectors are shown. Exon 23 of GLP, which encodes for amino acids 1028-1037 within the catalytic SET domain, was flanked with loxP sites in ES cells followed by routine procedure for generation of GLPfl/fl mice. Exons, unfilled rectangles; loxP sequences, filled triangles; FRT sequences (shaded octagons) that flank neo_R_ gene were removed from the targeted GLP locus in vivo by using FLP recombinase-transgenic mice. DTA, Diphtheria Toxin gene; TK, Thymidine Kinase gene; LAH/SAH, Long/Short Arm of Homology. Following BsrGI (B) digestion, DNA probe B was used to identify the targeted GLP locus. (B) Conditional neuron-specific ablation of GLP in the mouse forebrain. Postnatal, neuron-specific ablation of GLP in the mouse forebrain was achieved by breeding GLPfl/fl mice to _CamK2a_-Cre mice. Deficiency of GLP in neurons of the (1) cortex, (2) hippocampus and (3) striatum was confirmed by comparative immunohistochemical analysis of GLP protein expression in the forebrain of six-week old _Camk2a_-Cre; GLPfl/fl mice. Dotted lines indicate neuronal layer, arrowheads indicate examples of non-neuronal cells. (C) Reduced euchromatic H3K9me2 levels in GLP or G9a deficient neurons. Nuclear H3K9me2 distribution (red) in GLP (left panel) and G9a (right panel) deficient and control hippocampal neurons was analyzed by anti-H3K9me2 immunofluorescence; DNA was visualized by Draq5 staining (blue), diffuse blue staining indicates euchromatic DNA, punctate blue staining indicates heterochromatic DNA.

Figure 2

Figure 2. Deficiency in GLP or G9a alters gene expression in adult neurons

(A)Alteration of gene expression in GLP/G9a-deficient adult forebrain. Gene expression analysis was performed using normalized expression values from Affymetrix Mouse Genome 430 2.0 arrays. The gene expression levels in the striatum, hippocampus and hypothalamus of _CamK2a_-Cre; GLPfl/fl (n=4) and _CamK2a_-Cre; G9afl/fl (n=4) were compared to the respective gene expression values in control mice. The number of significantly >1.5 fold up- and down-regulated genes was determined using Welch t-test and Benjamin Hochberg procedure (p< 0.05). (B-D) Deficiency in GLP or G9a establishes a common pattern of altered gene expression. (B) The first three Venn diagrams show the number of genes with the significantly changed expression levels in GLP deficient (red) versus G9a deficient (blue) hippocampus, hypothalamus, and striatum. Overlapping genes for each specific brain region are shown in yellow and are compared in the fourth Venn diagram. (C, D) Each Venn diagram shows the number of genes with the significantly changed expression levels in GLP (C) or G9a (D) deficient hippocampus (red), hypothalamus (green) and striatum (blue). Expression levels of genes that are commonly affected by GLP (56 genes) or G9a (62 genes) deficiencies in all three brain regions (white) are shown as a heat map after hierarchical clustering was performed. Scale of gene expression goes from no detectable expression (blue) to the highest expression (red). Ingenuity pathway analysis has been used to assign these genes to a physiological process and the significantly enriched functions (p<0.05) with their corresponding genes are listed by significance.

Figure 3

Figure 3. Postnatal forebrain-specific GLP or G9a deficiency does not affect brain morphology or neuronal architecture

(A) The region-specific brain structures of twelve week-old Camk2a-Cre; GLPfl/fl (left panel), Camk2a-Cre; G9afl/fl (right panel) and control mice were analyzed using standard Nissl-staining. Representative images from saggital brain sections of the hippocampus, cortex and striatum are shown. (B-E) Individual GLP deficient and control Golgi-Cox stained granule cells in the dental gyrus of the hippocampus (B), medium spiny neurons in the striatum (C), and hippocampal pyramidal cells and their dendrites (D, E) were analyzed and representative images are shown.

Figure 4

Figure 4. G9a deficient Drd2 expressing medium spiny neurons (Drd2 MSNs) maintain Drd2 MSN-specific electrophysiological and morphological properties

(A) Drd2 MSNs (n=15 cells) in the striatum of Drd2-Cre; Drd2-eGFP; G9afl/fl mice responded to hyperpolarizing and depolarizing intrasomatic current steps in a prototypical fashion, displaying inward rectification and non-adapting trains of action potentials. (B) Drd2 MSNs lacking G9a retain Drd2 MSN-typical action potential waveforms, following brief, supra-threshold current injection. Inset: the rate of change of the membrane voltage/time plotted against voltage displays a normal action potential threshold. (C) Frequency-Current plot demonstrating a Drd2 MSN-typical pattern with a small but significant negative gain in excitability of Drd2 MSNs lacking G9a. Note: Markedly different frequency-current blot for closely related Dopamine 1 receptor (Drd1) MSN. (D) Reconstruction of biocytin-filled Drd2 MSNs revealed normal Drd2 MSN morphology in the absence of G9a. Wild-type Drd2 MSNs are shown in black, G9a deficient Drd2 MSNs are shown in red.

Figure 5

Figure 5. Deficiency of GLP/G9a results in reduced exploration and locomotor activity

(A-D) Reduced exploratory behavior in the absence of GLP/G9a in postnatal forebrain neurons. Open field analysis was used to measure locomotor activity and exploratory behavior. The distance traveled (cm) during 1 hr of open field analysis was determined for _Camk2a_-Cre; GLPfl/fl (n=21) (A) and _Camk2a_-Cre; G9afl/fl (n=12) (B) mice and is shown in 10 min bins. The total number of vertical episodes (C) and center/total distance ratio (D) during 1 hr in the open field are shown. The analysis included mice with combined GLP/G9a brain specific deficiencies (_Camk2a_-Cre; GLPfl/fl; G9afl/fl, n=8) as well as mice with brain specific ablation of the histone methyltransferase Ezh2 (_CamK2a_-Cre; Ezh2fl/fl, n=5). (E, F) Haploinsufficiency in GLP impairs locomotor activity and exploration. (E) The total distance traveled (cm) and (F) the total number of vertical episodes during 1 hr of open field analysis are shown for GLPfl/fl (n=22), Camk2a-Cre; GLP+/fl (n=18), Camk2a-Cre; GLPfl/fl (n=14), and GLP+/− (n=8) mice. Data are shown as means, error bars represent + s.e.m. *p<0.05, **p<0.01, ***p<0.001.

Figure 6

Figure 6. Deficiency of GLP/G9a in postnatal forebrain neurons results in complex behavioral abnormalities

(A) Unaltered locomotor function and balance in Camk2a-Cre; GLPfl/fl mice. Locomotor function and balance were studied using the accelerated rotarod analysis. The latency of Camk2a-Cre; GLPfl/fl and control mice (n=7 each) to fall off the rod (sec) during accelerated rotarod analysis for three consecutive days with 3 trials/day is shown. (B) Lack of awareness of potential danger in GLP and G9a deficient mice. The percentage of open arm entries, percentage of time spent in open arms and total arm entries during a 5 min period in an elevated plus maze are shown. The experimental groups included Camk2a_-Cre; GLP_fl/fl (n=18), Camk2a_-Cre; G9a_fl/fl (n=15) and respective control mice. (C) Deficits in motivation and reward in the absence of GLP/G9a. Sucrose preference test on _Camk2a_-Cre; GLPfl/fl, _Camk2a_-Cre; G9afl/fl and respective control mice (n=5 each) showed a decreased sucrose/water intake (ratio) in the absence of GLP/G9a. (D) Impaired learning and memory in the absence of GLP. The percentage of freezing responses for _Camk2a_-Cre; GLPfl/fl and their controls (n=13) during a standard contextual fear analysis are shown. Data are shown as means, error bars represent ± s.e.m. *p≤0.05, **p<0.01, ***p<0.001.

Figure 7

Figure 7. Selective deficiency of GLP/G9a in Drd1- or Drd2-expressing neurons alters the responsiveness to cell-type-specific stimuli

Open field analysis was used to assess the locomotor activity and exploratory behavior of _Drd1_-Cre; G9afl/fl (A, C, E, left column) and _Drd2_-Cre; G9afl/fl (B, D, F, right column) mice. To trigger Drd1 or Drd2 neuron-specific behavior, _Drd1_-Cre; G9afl/fl (n=10) (A, C, E), _Drd2_-Cre; G9afl/fl (n=9) (B, D, F), and littermate control mice were treated with the Drd1-agonist SKF 81297 or the A2a-antagonist caffeine, respectively. (A, B) The distance travelled (cm) during open field analysis is shown. Data are shown in 5 min bins; consecutive conditions from left to right are: 20 min exposure to new environment, 20 min post saline injection, and 30 min post Drd1-agonist SKF 81297 (5mg/kg ip) injection (A), or 45 min post A2a-antagonist caffeine (10 mg/kg ip) injections (B). (C, D) The total distance travelled (cm) for each of the three conditions is shown. (E, F) The total number of vertical episodes during each of the three conditions is shown. G9a deficient mice are shown in light gray, littermate controls in dark gray; Data are shown as means, error bars represent ± s.e.m. **p<0.01, ***p<0.001.

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