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Setdb1-mediated histone H3K9 hypermethylation in neurons worsens the neurological phenotype of Mecp2-deficient mice - PubMed

Setdb1-mediated histone H3K9 hypermethylation in neurons worsens the neurological phenotype of Mecp2-deficient mice

Yan Jiang et al. Neuropharmacology. 2011 Jun.

Abstract

Rett syndrome (RTT, OMIM # 312750), a neurodevelopmental disorder of early childhood, is primarily caused by mutations in the gene encoding methyl-CpG-binding protein 2 (MECP2). Various molecular functions have been ascribed to MECP2, including the regulation of histone modifications associated with repressive chromatin remodeling, but the role of these mechanisms for the pathophysiology of RTT remains unclear. Here, we explore whether or not neuronal expression of the histone H3-lysine 9 specific methyl-transferase, Setdb1 (Set domain, bifurcated 1)/Eset/Kmt1e, which is normally present only at low levels in differentiated neurons, rescues the RTT-like phenotype of Mecp2-deficient mice. A myc-tagged Setdb1 cDNA was expressed through the tau locus for ubiquitous expression in CNS neurons, or under control of the calcium/calmodulin-dependent protein kinase II (CK) promoter to selectively target postmitotic neurons in forebrain. However, the CK-Setdb1 transgene lead to an enhanced neurological deficit, and the tauSetdb1 allele further shortened life span of mice with a brain-wide deletion of Mecp2 during prenatal development. In contrast, no neurological deficits or premature death was observed in CK-Setdb1 and tauSetdb1 mice expressing wildtype Mecp2. However, levels of trimethylated H3K9 at pericentromeric repeats were fully maintained in differentiated neurons from symptomatic Mecp2 null mutant mice. Based on these results, we draw two conclusions: First, neuronal chromatin in RTT brain is not affected by a generalized deficit in H3K9 trimethylation. Second, artificial up-regulation of this repressive chromatin mark via Setdb1 gene delivery specifically to neurons is harmful for the Mecp2-deficient brain. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

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Figures

Figure 1
Figure 1. Setdb1 expression is down-regulated during the course of neuronal maturation

A, Representative immunoblots showing Setdb1 (upper band), and for loading control, histone H4 (modification-independent) (lower band) immunoreactivity from E15.5 cortical plate (lanes 1, 2) and adult (postnatal week 8–12) cerebral cortex (lane 3). Band in lane 4 is same as in lane 3, shown again after a longer film exposure. 5 μg (lane 1) or 20 μg (lane 2, 3) of protein was loaded. B, Bar graphs showing relative Setdb1 mRNA levels as determined by qRT-PCR in mouse cerebral cortex at different stages as indicated: embryonic day (E) 12.5, 15.5, 18.5, postnatal (P) 7, and adult (8 weeks and older). Data are expressed as mean ± SEM after normalization to 18S rRNA. N = 5/age group. C, Representative Setdb1 immunoblots from cerebral cortex at different stages as indicated. Data are expressed as mean ± SEM after normalization to GADPH. N = 4/age group. *p<0.05, #p<0.01 (B, C) as compared to E 12.5, one-way ANOVA. Notice that both Setdb1 mRNA and protein decline during the course of cortical maturation. D–F, Primary cultures of cortical neurons from E.15.5 mouse cortical plate. D, Immunofluorescence of TuJ1 neuronal marker (red), GFAP (green) and DAPI (blue) at DIV 4. E–F, Representative blots (top) and bar graphs (bottom) showing Setdb1 (E) and MeCP2 (F) protein levels at DIV 2, 7, and 14. Data are expressed as mean ± SEM after normalization to H4. Notice opposing patterns of expression over time, as recognized by decline in Setdb1 and increase in MeCP2 expression. N = 4/age group. *p<0.05, #p<0.01 as compared to DIV 2, one-way ANOVA.

Figure 2
Figure 2. Widespread Setdb1 expression in the developing mouse brain

Representative images showing Setdb1 mRNA expression in coronal sections from frozen (A, B) or paraffin-embedded (C) tissue blocks at E12.5 (A) or E15.5 (B, C). Sections were hybridized with digoxigenin-labeled anti-sense (a) and sense (b) Setdb1 RNA probes. Notice robust expression in all compartments of the developing brain. Abbreviations: CI: cerebral cortex, layer 1, CP: caudoputamen, CxP: cortical plate, cpn: caudate putamen neuroepithelium, GP: globus pallidus, H: hippocampus, iz: intermediate zone, LV: lateral ventricle, ncn: neocortical neuroepithelium, SP: subplate, svz: subventricular zone (striatum), svzc: subventricular zone (neocortex), V3: third ventricle, nc: neocortical plate, iz: intermediate zone, vz: ventricular zone. Scale bars: 200 μm (A, B), 50 μm (C).

Figure 3
Figure 3. CK-Setdb1 worsens the RTT phenotype of Mecp2 mutant mice

A, Body weight (at 4 weeks of age); B, locomotor activity (8 weeks of age); and C, rotarod performance (11 weeks of age) of wildtype, CK-Setdb1, Mecp2−/y and CK-Setdb1/Mecp2−/y. N=23–31/genotype (locomotor activity), N=13–16/genotype (rotarod). *p<0.05 (Mecp2−/y), #p<0.01 (CK-Setdb1/Mecp2−/y) as compared to wildtype; *p<0.05 (CK-Setdb1/Mecp2−/y) as compared to Mecp2−/y, one-way ANOVA. D, Kaplan-Meier survival curves. N=19–24/genotype. Notice worsening of neurological deficits (A–C) and no prolonged survival (D) of CK-Setdb1/Mecp2−/y mice, as compared to Mecp2−/y.

Figure 4
Figure 4. tauSetdb1 mice

A, Targeting vector, including myc-tagged Setdb1 cDNA (myc-Setdb1), neomycin resistance cassette (NEOR), and homologous flanking sequences of Mapt (tau) (gray box). Insertion of myc-Setdb1-NEOR cassette at tau exon 2 ATG start (marked by vertical line). Locations of 5′-external (1), internal (2) and 3′-external (3) probes used for Southern blot analysis as indicated. B: BamHI; E: EcoRI. B, Representative images showing Southern blot analysis of WT and targeted embryonic stem cell clones (F1, D3, G11, E12) after digestion with BamHI or EcoRI. a) Hybridization with the internal probe (2) results in a 9887-bp band for the targeted allele (ki). b) Hybridization with the 5′-external probe (1) results in a 9175-bp WT band and a 4432-bp band for the targeted allele. c) Hybridization with the 3′-external probe (3) results in a 9175-bp WT band and a 6768-bp band for the targeted allele. C, Immunohistochemical stains with anti-myc antibody, notice labeling of neuronal nuclei in cerebral cortex (a), striatum (c), hippocampus (e) and cerebellum (g) of tauSetdb1 mice; (b, d, f, h) show corresponding sections from wildtype controls. Scale bar, 10 μm. D, Bar graphs showing Setdb1 mRNA expression in cerebral cortex (a) and striatum (b) from wildtype and tauSetdb1 mice. N=3–4/genotype. #p<0.01, Mann Whitney test. E, Anti-myc immunoblot showing expected 180kDa band in cerebral cortex (CC), striatum (Str), hippocampus (Hipp), cerebellum (Cbl), thalamus (Thal), hypothalamus (Hypo), periaqueductal gray (PAG), and substantia nigra (SN) in adult tauSetdb1 (+), but not in WT mice (−).

Figure 5
Figure 5. tauSetdb1 accelerates premature death of Mecp2 deficient mice

A, Rotarod performance from wildtype, tauSetdb1, Mecp2−/y, and tauSetdb1/Mecp2−/y at 12 weeks of age. N=9–17/genotype. *p<0.05 as compared to Control, one-way ANOVA. B, Kaplan-Meier survival curves. N=11–25/genotype. The group of tauSetdb1/Mecp2−/y mice was comprised of 8 hetero- and 17 homozygous tauSetdb1 knock-in mice. Notice left shift in survival curve of tauSetdb1/Mecp2−/y mutant mice (Kaplan-Meier, P<0.07). Notice further no significant effect of tauSetdb1 on rotarod performance.

Figure 6
Figure 6. H3K9 tri-methylation is normal in neuronal heterochromatin of the Mecp2 deficient brain

A, Representative image showing immunofluorescence of MeCP2/DAPI counterstain (a/b, c/d, green/blue), and NeuN/DAPI (e/f, g/h, green/blue) in nuclei after hypotonic extraction from adult Mecp22lox/y/Nestin-cre conditional knockout (Mecp2−/y) (a/b, e/f) and wildtype (c/d, g/h) forebrains. Notice lack of MeCP2 immunoreactivity in the mutants. Scale bar, 10 μm. B, Representative FACS scatter plots from (a) negative control (NeuN−) processed without NeuN antibody, and (b) sample processed with NeuN antibody (NeuN+). Only the population of nuclei in sector P3, showing robust immunofluorescence, were collected during FACS. C, Chromatin of FACS-sorted NeuN+ nuclei from wildtype and Mecp2−/y were subjected to anti-H3K9me3 ChIP, and then processed together with input by slot-blot hybridized to major satellite DNA probe. Bar graph showings no changes in H3K9me3 levels in pericentromeric heterochromatin of neuronal nuclei extracted from 8 weeks old wildtype and Mecp2−/y mice. N=8/genotype. Data shown as mean ± SEM.

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