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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 mechanism for the pathophysiology of RTT remains unclear. Here, we explore whether or not neuronal expression of the histone H3-lysine 9 specific methyltransferase, 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.
RTT (MIM 312750) is a neurodevelopmental disorder in which affected girls show developmental regression often after a 6–18 month symptom-free period, while boys typically do not survive beyond early postnatal life (Chahrour and Zoghbi, 2007). The majority of cases are due to mutations in the X-linked gene, methyl-CpG-binding protein 2 (MECP2). Presently, treatment options for RTT patients address only the symptoms (Percy, 2002), while effective prevention and cure will require a better understanding of the underlying molecular pathology. Important clues have been provided by studies on genetically engineered mice. A Rett-like phenotype is induced by conditional ablation of Mecp2 in neurons (Chen et al., 2001; Guy et al., 2001) and rescued with pan-neuronal expression of a Mecp2 cDNA (Luikenhuis et al., 2004). These findings underscore that RTT is a neuronal disease. Importantly, activation of Mecp2 expression—even after mutant mice became symptomatic—resulted in a robust phenotypic reversal, which suggested that the neurological deficits in this disorder may not be irreversible (Guy et al., 2007). It remains to be determined, however, which of the various molecular actions ascribed to MeCP2—including remodeling of facultative or constitutive heterochromatin, as well as a more complex role in the regulation of gene transcription, mRNA splicing, and structuring of higher order chromatin (Chahrour et al., 2008; Chahrour and Zoghbi, 2007; Jordan et al., 2007; Kumar et al., 2008; Skene et al., 2010; Yasui et al., 2007)—are critical for the maintenance of neuronal health and the development of RTT.
Here, we test the hypothesis whether repressive chromatin modifications, specifically, tri-methylation of histone H3-lysine 9 (H3K9me3), could rescue the phenotype of Mecp2 deficient mice. To investigate this, we engineered mice with neuron-specific expression of the H3K9 specific methyltransferase, SET domain, bifurcated 1 (Setdb1, also known as Eset, Kmt1e) (Schultz et al., 2002; Yang et al., 2002). Setdb1 contains, in addition to a SET and a Tudor domain, a methyl-CpG-binding domain (Blackburn et al., 2003; Schultz et al., 2002; Yang et al., 2002), and, like MeCP2 (Jones et al., 1998; Nan et al., 1998), interacts with the mSin3-histone deacetylase (HDAC) co-repressor complex (Yang et al., 2003). Although MeCP2, in contrast to Setdb1, has no methyltransferase activity, it regulates H3K9 methylation via its interaction with Suppressor of variegation 3–9 homolog 1 (Suv39H1) methyl-transferase (Lunyak et al., 2002). Therefore, both Setdb1 and MeCP2 are implicated in repressive chromatin remodeling, but potentially involve different protein complexes.
In the present study, we observed that expression of MeCP2 and Setdb1 undergo dynamic changes during brain development, but in a strikingly opposite manner. Thus, Setdb1 levels became down-regulated, while Mecp2 was up-regulated in mature neurons, consistent with earlier reports(Akbarian et al., 2001; Balmer et al., 2003; Jung et al., 2003; Shahbazian et al., 2002b). Given that (as discussed above) both Mecp2 and Setdb1 contain a mCpG binding domain and are involved in repressive chromatin remodeling, suggesting a possible overlap in function, we therefore asked whether a persistent up-regulation of Setdb1 could rescue Mecp2 deficient mice that typically develop symptoms several weeks after birth (Chen et al., 2001; Guy et al., 2001; Shahbazian et al., 2002a). To address this question, we used two independent mouse lines to express Setdb1 in differentiated neurons: either by a transgene driven by the calcium/calmodulin-dependent protein kinase II (CK) promoter, or through the endogenous tau locus.
All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School (UMASSMED). Animals were housed in groups of 2–4 per cage with food and water ad libitum under 12-hour light/dark cycle.
The PGKNeo resistant selectable marker from plasmid pK-11 (Frt-PGKNeo-Frt-Loxp-pBSSK) (Meyers et al., 1998) was inserted into the modified pCS2-MT-ESET (a gift from Dr. Liu Yang, University of Arkansas for Medical Sciences) (Yang et al., 2003). Then, the myc-Setdb1 cDNA was fused in-frame into the exon 2 of mouse Mapt (tau) locus, using pTau-KR plasmid containing 3.8 kb of tau genomic sequence (a gift from Dr. Rudolf Jaenisch, Whitehead Institute, Cambridge, MA) (Luikenhuis et al., 2004). For in-frame fusion, an adaptor (5′-ATCGATGGATATC-3′) with ClaI and EcoRV sites was introduced into pTau-KR, replacing a 14 bp fragment and ATG start codon of wildtype tau exon 2 (5′-ATGGCTGACCCTCG-3′). The targeting vector, pTau-MT-ESET, was linearized with SacII and electroporated into 129Sv-strain AB2.2 embryonic stem (ES) cell lines at the Transgenic Animal Modeling Core. Neomycin-resistant clones were first pre-screened by PCR for correct insertion and further assayed by Southern blot with internal and external probes (see Results). The 5′ external probe was comprised of 447 bp fragment (subcloned from PCR product using wildtype AB2.2 ES cell DNA) located 2 kb upstream from the ATG condon of wildtype tau locus. The 3′ external probe was comprised of a subcloned, 982 bp fragment from wildtype ES cell DNA, located 4.7 kb downstream from the ATG codon of wildtype tau. The internal probe was a 1247 bp EcoRI/BamHI fragment from the 5′-end of Setdb1 cDNA. Suitable clones (63F1, 63D3, 63E12) were used to generate chimeras by injection into C57BL/6 blastocysts at the Transgenic Animal Modeling Core. Chimeras were mated to C57BL/6J females; germline transmission was achieved with clone #63D3. tauSetdb1 was then crossed into the conditional Mecp2 knockout for the rescue experiments.
For the rescue experiments, only male mice were included in this study. CK-Setdb1 transgene: To obtain CK-Setdb1/Mecp2−/y rescue mice, CK-Setdb1 transgenic males (Jiang et al., 2010) were bred with Mecp2 2lox/2lox females. Then, CK-Setdb1+/o/Mecp2 2lox/2lox females were crossed with Nestin-Cre+/o transgenic males, and the following 4 genotypes were obtained at the expected frequencies: (1.) Mecp22lox/y (for simplicity, termed “wildtype” in the following), (2.) CK-Setdb1+/o/Mecp22lox/y (for simplicity, termed “CK-Setdb1” in the following), (3.) Nestin-Cre+/o/Mecp22lox/y (for simplicity, termed “Mecp2 −/y” in the following), and (4.) CK-Setdb1+/o/Nestin-Cre+/o/Mecp22lox/y (for simplicity, termed “CK-Setdb1/Mecp2−/y” in the following). After Cre-mediated ablation of Mecp2 coding sequences in this line of mutant mice, MeCP2 immunoreactivity becomes non-detectable, as previously described (Chen et al., 2001) (Fig. 6).
Previous studies found that tau knockout mice, or animals homozygous for various tau knock-in alleles, which effectively do not express wildtype tau protein, appear in good health and without overt neurological defects (Dawson et al., 2001; Denk and Wade-Martins, 2007; Tucker et al., 2001). Therefore, both heterozygous and homozygous tauSetdb1 mice were included in this study. TauSetdb1+/o/Mecp2 2lox/2lox females were crossed with tauSetdb1+/o/Nestin-Cre+/o males, and the following genotypes were obtained at the expected frequencies: (1.) Mecp22lox/y (for simplicity, termed “wildtype” in the following), (2.) tauSetdb1/Mecp22lox/y (for simplicity, “tauSetdb1” in the following refers to both hetero- and homozygous knock-in animals), (3.) Nestin-Cre+/o/Mecp22lox/y (for simplicity, termed “Mecp2−/y” in the following), and (4.) tauSetdb1/Nestin-Cre+/o/Mecp22lox/y (for simplicity, termed “tauSetdb1/Mecp2−/y” in the following).
Locomotion was monitored in a rectangular test chamber (19.1×29.2×12.7cm) with clean bedding, using a photocell beam based computer detecting system (San Diego Instruments). Mice were placed into the test chamber for 90 minutes under standard room lighting conditions. The beam breaks were collected every 5 minutes to evaluate the spontaneous locomotor activity.
Rota-Rod (UGO BASILE 47600) was used to measure motor coordination in mice. The rotarod was set up with a slow acceleration mode, in which the rotation speed is accelerated from 2 to 40 rpm in 5 minutes. During the training session, mice were subjected to 10 consecutive 5-min trials with a 5-min intertrial interval. The latency (sec) to falling off or, alternatively, making a full-turn rotation was recorded. 24 hours after training, mice were tested by receiving 3 consecutive trials (at 5 min intervals), and the mean latency was used to present the performance.
Entire heads (Embronic day (E) 12.5), or whole brains (E15.5) were fixed with 4% phosphate-buffered paraformaldehyde (4% PFA) at 4°C overnight, then frozen or paraffin-embedded for subsequent in situ hybridization. 18 μm sections from frozen samples, or 4 μm from paraffin-embedded samples were hybridized to 50 ng/50 μl of DIG-11-UTP labeled sense or antisense Setdb1 RNA probes at 60°C overnight, and then washed and digested with RNase A, and developed with the DIG Nuclei Acid detection kit (Roche, Indianapolis, IN), according to the manufacturer’s instructions. The Setdb1 probe was in vitro transcribed and was corresponding to a 347 bp fragment in 3′-end of Setdb1 cDNA. Similar studies were conducted on brains sections prepared from perfusion-fixed adult animals.
Cerebral cortex was dissected from C57Bl6/J mice at E15.5 and dissociated by trypsin digestion and trituration as described (Lesuisse and Martin, 2002). Cells (1.5×106 cells/well) were plated in poly-l-lysine coated 6-well plates, cultured in Neurobasal medium (Life Technologies) supplemented with B27 (GIBCO, #17504-044), 200 mM L-glutamine, and 1% v/v penicillin and streptomycin (GIBCO, #15140). Antimitotic reagents, Uridine 5′-Triphosphate and 5-Fluoro-2-deoxy Uridine (10 mg/ml each) (Sigma) were added to limit glia proliferation. Cells were harvested after 2, 7, or 14 days cultured in vitro (DIV), and then prepared for Western blot or immunofluorescence staining.
Cultured cells were fixed with phosphate-buffered 4% PFA, permeabilized with 0.2% Triton X-100, blocked with 10% goat serum, and then incubated with primary antibody (anti- TuJI, -GFAP) overnight at 4 °C. The signal was detected by Alexa 488- or 594-conjugated secondary antibodies. Nuclei isolated from tissue homogenates in hypotonic solution (see below) were fixed in phosphate-buffered 4% PFA for 5 min, spread on slides and airdried, and then stained for anti-MeCP2 (Sigma, mouse monoclonal, clone men-8, directed against the N-terminus of MeCP2) and anti-NeuN immunofluorescence (Chemicon).
Samples were homogenized directly in 1x Laemmli buffer with 1x Complete Proteinase Inhibitor (Roche Applied Science, #11697498001), incubated at 37°C for 10 min, and centrifuged at 13,500 g for 5 min at 4°C. The s upernatant was denatured at 95°C for 5 min, electrophoresed on a 4%–20% linear gradient Tris-HCl gel (Bio-Rad, Hercules, California), and then transferred to nitrocellulose membrane (Bio-Rad, 0.4 μm pore size). Immunostainings were performed with anti-c-myc (A-14) (Santa Cruz, #sc-789), anti-Setdb1 antibody (Upstate, #07-378), and anti-MeCP2 (Upstate #07-013, rabbit polyclonal, against MeCP2 C-terminus) and, for loading controls, anti-GADPH (Abcam, #ab9485) and anti-histone H4 antibody (Upstate, #07-108). Immunoreactivity was detected with peroxidase-conjugated secondary antibody in conjunction with chemiluminesence-based film autoradiography (Super Signal West Dura Extend Reagent; Pierce, Rockford, Illinois). For quantification, Quantity-One (Biorad) software was used.
Total RNA was extracted from cerebral cortex, striatum, or forebrain of mice brains using RNeasy Lipid Tissue Mini kit (QIAGEN, #74804) according to manufactor’s protocol, and then subjected to SYBR green based one-step RT-PCR using Taqman One-Step RT-PCR Mix Reagents (Roche, #4309169). Two different sets of primers were designed to amplify approx. 100 bp intron-spanning fragments in the 3′-end of Setdb1 cDNA. The primers were, 18-2: left 5′-CTTCGCCGACCAGCTAGTAA-3′, right 5′-GTTCCACTTTCCCCCTCACT-3′; 19-2: left 5′-CTTCTGGCTCTGACGGTGAT-3′, right 5′-GGAAGCCATGTTGGTTGATT-3′. The Bdnf primers used were: left 5′-GCGCCCATGAAAGAAGTAAA-3′, right 5′-TCGTCAGACCTCTCGAACCT-3′. Quantifications were done with 18S rRNA as an internal control (Schroeder et al., 2007).
Nuclei were extracted from adult mouse forebrain, immunotagged with anti-NeuN, and sorted as described (Jiang et al., 2008). Using this procedure, background immunofluorescence was minimal, and all sorted samples had a purity of 95% (NeuN+ nuclei) or more.
Samples were prepared either by micrococcal nuclease (MNase) digestion for native chromatin immunoprecipitation (NChIP) as described (Huang et al., 2006), or fixed by 1% formalin, sheared by sonication (Branson Sonifier 250) for XChIP. NChIP: In brief, MNase digestion was performed with a working concentration of 4U/mL at 37°C for 5 min. The resulting mono-nucleosomal prep aration (approx. 146 bp) was precleaned by incubating with protein G agarose (which should also remove the antibodies used for NeuN labeling of nuclei), and then subjected to immunoprecipitation using anti-trimethyl-H3K9 (H3K9me3) antibody (Upstate, #07-442). Typically, we used 6 – 8 ×106 FACS-sorted nuclei as input. XChIP: Samples were fixed in 1% formalin for 5 min at room temperature, sonicated at power level 6 (Branson, Danbury CT) in ice water (10 runs of 30 sec impulse with 1 min resting interval), pre-cleaned, and then subjected to H3K9me3 immunoprecipitation. Control samples included samples processed with normal rabbit IgG, in parallel to samples with specific antibodies. Input DNA and immunoprecipitated DNA were subjected to slot-blot hybridization using a 32p labeled oligonucleotide probe 5′ GGACC TGGAA TATGGC GAGAA A 3′ to label mouse major satellite DNA.
To examine whether Setdb1 is developmentally regulated in mouse brain, we monitored Setdb1 expression in embryonic, postnatal and adult cerebral cortex (Fig. 1A–C). Levels of Setdb1 protein (Fig. 1A, C) and mRNA (Fig. 1B) were highest at the earliest stage examined (E12.5), but then showed a progressive and significant decline, resulting in 8-fold lower Setdb1 immunoreactivity in 8–12 week old (adult) cortex (Fig. 1C). Although Setdb1 was subject to developmental downregulation, the protein was readily detectable in immunoblots from mature brain (Fig. 1C). To confirm that Setdb1 is expressed throughout all regions of the developing brain, we conducted in situ hybridization on E12.5 and E15.5 brains. The Setdb1 antisense probe resulted in robust labeling throughout the developing brain (Fig. 2A, B), including mitotic cells within proliferative layers, such as the ventricular zone, as well as postmitotic neurons residing in the cortical plate and other territories (Fig. 2B, C). In contrast, labeling of sections from adult brain was weak and often not distinguishable from background (data not shown).
To further test whether the observed downregulation of Setdb1 protein during development (Fig. 1A, C) occurs within differentiating neurons, we monitored the temporal pattern of Setdb1 expression in primary neuronal cultures derived from E15.5 mouse cortical plate (Fig. 1D). After 14 days of culture in vitro, Setdb1 immunoreactivity was significantly decreased by more than 2-fold (Fig. 1E). In contrast, during the same time period, levels of MeCP2 immunoreactivity showed a 14-fold, significant increase (Fig. 1F). The latter finding is in good agreement with earlier studies in rodent (Jung et al., 2003; Shahbazian et al., 2002b), monkey (Akbarian et al., 2001) and human brain (Shahbazian et al., 2002b) reporting that MeCP2 levels are up-regulated during the course of neuronal maturation. We conclude that expression of both Setdb1 and MeCP2 undergo dynamic changes in differentiating neurons of developing brain, though in a strikingly opposite manner. This observation, in view of the potential overlap in MeCP2 and Setdb1 function, led us to speculate that the RTT phenotype in adult Mecp2 deficient mice could be ameliorated by artificial expression of Setdb1 in mature neurons.
The calcium/calmodulin-dependent protein kinase II (CK) promoter typically drives transgene expression in a substantial portion of neurons, primarily those located in the forebrain (Mayford et al., 1996). The CK promoter-regulated, conditional or inducible expression of MeCP2 transgene significantly delays the onset of neurological symptoms and the premature death of Mecp2 deficient mice (Giacometti et al., 2007; Jugloff et al., 2008), although negative findings have also been reported (Alvarez-Saavedra et al., 2007). Therefore, we wanted to find out if CK-driven Setdb1 expression would affect the phenotype of Mecp2 deficient mice. To this end, we generated CK-Setdb1 transgenic mice, in which a myc-tagged full length Setdb1 cDNA was under the control of the CK promoter. Significant increases of Setdb1 expression were detected in most regions of adult CK-Setdb1 transgenic forebrain. This was associated with increased of H3K9 methyl-transferase activity, because there was a significant, approximately 60% increase in major satellite DNA-associated trimethyl-H3K9 (H3K9me3) in CK-Setdb1 forebrain (Jiang et al., 2010). The increase in satellite repeat-associated H3K9 methylation was specific because global tissue levels of H3K9 methylation, when measured in brain homogenates by immunoblot, remained unchanged in CK-Setdb1 forebrain (Jiang et al., 2010). The observation that changes in Setdb1 levels are not associated with altered H3K9 methylation overall is not too surprising, however, given that there are genome-wide at least 6 or 7 H3K9 specific methyltransferase enzymes (Kouzarides, 2007), and many of these are robustly expressed in adult brain (Lein et al., 2007).
Because the disease progression and neuronal alterations of Mecp2 deficient mice are known to be significantly affected by levels of brain-derived neurotrophic factor (Bdnf) (Chang et al., 2006; Wang et al., 2006; Zhou et al., 2006), we examined if expression of this neurotrophin was altered in the forebrain of adult CK-Setdb1 transgenic mice. Levels of Bdnf mRNA, when normalized to 18S rRNA transcript, were not significantly different between transgenic and littermate controls (mean ± S.E.M., wildtype: 5.69 ± 0.59; CK-Setdb1: 5.37 ± 0.39, N =6/group). Next, we crossed the CK-Setdb1 transgenic line with Mecp2 conditional mutant mice for a Cre/loxP mediated deletion of the methyl-CpG binding domain (MBD) (Chen et al., 2001), which is encoded by exon 2 of Mecp2 isoform 1, or exon 3 of isoform 2, respectively (Kriaucionis and Bird, 2004; Mnatzakanian et al., 2004). As expected (Chen et al., 2001), conditional, Nestin-Cre mediated deletion of Mecp2 during prenatal brain development resulted in premature death beginning at 2 months of age, impaired motor coordination, and decreased locomotor activity (Fig. 3B–D). Furthermore, the Mecp2 deficient mice, which are of a mixed genetic background that includes C57Bl6/J, were significantly underweight at 4 weeks of age (Fig. 3A), as previously reported for the mutants of that inbred line (Guy et al., 2001). At later ages, mutant weight became more variable (data not shown). Survival, motor activity and coordination, and body weight of CK-Setdb1 transgenic mice expressing wildtype MeCP2 were not significantly different from controls (Fig. 3). However, the transgene did not rescue the decrease in body weight, nor did it prevent neurological symptoms and premature death of Mecp2 mutant mice (Fig. 3). Instead, a pronounced deficit in novelty-induced locomotion was observed in CK-Setdb1/Mecp2−/y mice, which showed a significant 40%, or 20% decrease in activity scores compared to wildtype controls, or Mecp2−/y animals, respectively (Fig. 3B).
As described above, neuronal expression of Setdb1 in adult forebrain does not rescue the Rett-like phenotype of Mecp2 mutant mice. Of note, previous studies that utilized the CK promoter to rescue Mecp2 deficient mice yielded variable results (Alvarez-Saavedra et al., 2007; Giacometti et al., 2007; Jugloff et al., 2008). In contrast, when Mecp2 cDNA is expressed under control of the promoter of the microtubule-binding protein tau, complete rescue is achieved (Luikenhuis et al., 2004). This may be due to the fact that expression through the endogenous tau locus is pan-neuronal, starting during prenatal development and continues throughout all postnatal stages and adulthood (Luikenhuis et al., 2004). Another advantage of the tau locus is that mice not expressing wildtype tau protein appear in good health and without overt neurological defects (Dawson et al., 2001; Denk and Wade-Martins, 2007; Harada et al., 1994; Tucker et al., 2001) other than subtle defects in motor coordination and fear conditioning in some studies (Denk and Wade-Martins, 2007).
To express Setdb1 through the endogenous tau locus, we fused the myc-tagged Setdb1 cDNA first with a neomycin resistance cassette, and then ligated it into a tau targeting vector as previously described (Luikenhuis et al., 2004), replacing the ATG start codon of wildtype tau and surrounding sequences (Fig. 4A). The targeting construct was electroporated into AB2.2 embyronic stem cells. From 384 neomycin-resistant clones, 13 were targeted correctly, including clone #63D3 (Fig. 4B) that was passed through the germline to generate tauSetdb1 mice. As expected, expression of 180 kDa myc-tagged Setdb1 in these knock-in mice was neuron-specific and present in all brain regions examined (Fig. 4C–E). As mentioned above, double-banding of the 180kDa immunoreactivity was observed at shorter exposure times (Fig. 4E).
Next, we crossed the tauSetdb1 knock-in with Mecp2 conditional mutant mice. Similar to the experiments described above, the Mecp2 deficient mice of this outbred colony also showed significant deficits in motor coordination assayed by rotarod (Fig. 5A) and premature death starting around 8 weeks of age (Fig. 5B). However, in this colony, the body weight of Mecp2 mutant was not different from controls (<2%, data not shown). Furthermore, the survival and motor coordination of tauSetdb1 knock-in mice expressing wildtype Mecp2 were not significantly different from controls (Fig. 5A). However, the tauSetdb1 allele conferred a strong trend (Kaplan-Meier, p < 0.07) for further acceleration of premature death in Mecp2 mutant mice (Fig. 5B). This Rett-like phenotype was observed in both hetero- and homozygous tauSetdb1 knock-in mice (Fig. 6B legend and data not shown). Thus, neither the CK-Setdb1 transgene nor the tauSetdb1 knock-in allele ameliorated the symptoms induced by Mecp2 deficiency.
As mentioned, transgene-derived expression of myc-Setdb1 cDNA resulted in H3K9 hyper-tri-methylation at pericentric repeats. Of note, MeCP2 is highly concentrated in pericentromeric heterochromatin (Lewis et al., 1992). In the mouse, pericentric heterochromatin is mainly defined by tandem repeats of A/T-rich major satellite repeats comprised of approximately 105 copies of a 234 base-pair unit (Waterston et al., 2002). Among the various repressive histone methylation marks, H3K9me3 is highly and consistently enriched at sites of major satellite repeats in different cell types (Martens et al., 2005). Importantly, immunoprecipitation studies revealed association of MeCP2 with Suppressor of variegation 3–9 homolog 1 (Suv39H1) (Lunyak et al., 2002), a histone methyl-transferase essential for H3K9 trimethylation at pericentric repeats (Peters et al., 2001). Together, these findings imply that MeCP2, bound to the methylated CpG’s of the major satellite, regulates H3K9me3 at these repeats via an interaction with Suv39H1. Therefore, loss of MeCP2 could affect orderly methylation of H3K9 in chromatin surrounding major satellite repeats. However, until now this hypothesis has not been tested in Mecp2 deficient brain. Importantly, MeCP2 is localized almost exclusively in mature neurons (Akbarian et al., 2001; Chen et al., 2001; Jung et al., 2003; LaSalle et al., 2001; Shahbazian et al., 2002b), which reside in brain together with glia and other cells mostly in a 2:1 – 1:2 ratio (Herculano-Houzel et al., 2006; Sherwood et al., 2006); therefore, a conclusive assessment of MeCP2 association with neuronal heterochromatin would require separation from non-neuronal elements. Here, we extracted neuronal nuclei from whole forebrain of 8 week old Mecp2−/y and male controls; at this stage, the Mecp2 mutant mice already are symptomatic and show deficits in locomotor activity (Fig. 3B). The neuronal nuclei were sorted via FACS after immunotagging with anti-NeuN antibody (Fig. 6A, B), employing a previously described protocol (Jiang et al., 2008). Subsequently, neuronal nuclei were subjected to digestion with micrococcal nuclease and, after ChIP with anti-H3K9me3 antibody, the mono-nucleosomal DNA from immunoprecipitates, input, and no antibody control samples were blotted without prior amplification. Overall DNA yields of input samples were similar between mutant and control samples, indicating that the nucleosomal organization of the genome is grossly maintained in the absence of MeCP2 (data not shown). Furthermore, levels of H3K9me3 at major satellite repeats were completely preserved in neurons from Mecp2 mutant brain, compared to wildtype controls (Fig. 6C). We conclude that MeCP2 is not essential for the establishment or maintenance of the H3K9me3 mark at major satellite DNA in neuronal nuclei.
Using genetically engineered mice, we demonstrate that persistent up-regulation of neuronal Setdb1 expression in adult brain results in hypertrimethylation of H3K9 at pericentromeric repeat DNA, and worsens the phenotype of Mecp2 deficient mice. Furthermore, we show that H3K9 trimethylation levels remain unaltered in pericentromeric chromatin of neuronal nuclei from symptomatic Mecp2 deficient mice. Therefore, loss of Mecp2 is not associated with a generalized deficit in H3K9 tri-methylation at DNA repeats in diseased neurons. These negative findings of the present study, however, do not rule out that other types of histone modifications show global alterations in the Mecp2 deficient neurons. Indeed, a recent study reported that chromatin of mature Mecp2−/− neurons is affected by a generalized increase in histone acetylation (Skene et al., 2010).
The finding reported here—normal levels of trimethylated H3K9 in pericentric heterochromatin of Mecp2 deficient neurons— is in good agreement with recent findings that methylation of H3K9 and other histone residues are maintained at normal levels (or show only minimal changes) in bulk chromatin extracted from whole tissue of selected brain regions of Mecp2 null mutant mice (Urdinguio et al., 2007) or peripheral blood cells of RTT patients (Kaufmann et al., 2005). On the other hand, a study utilizing quantitative immunofluorescence for H3K9me3 found a generalized decrease in Mecp2 deficient neuronal nuclei, although the punctate staining of heterochromatic foci was preserved in the mutants (Thatcher and LaSalle, 2006). Likewise, no consistent changes in global histone acetylation were reported in Mecp2/MECP2 deficient brain (Shahbazian et al., 2002a; Thatcher and LaSalle, 2006; Urdinguio et al., 2007), blood cells, or cell lines (Balmer et al., 2002; Kaufmann et al., 2005; Wan et al., 2001), and it remains possible that this variation may be in part due to differences in the genetic background and type of Mecp2/MECP2 mutations, or even the specific histone molecules or residues examined.
In the present study, genetically engineered mice with a sustained up-regulation of Setdb1 expression in adult brain displayed hyper-trimethylation of H3K9 in neuronal chromatin, yet did not develop an overt neurological phenotype. However, in the Mecp2 mutant mice, the CK-Setdb1 transgene resulted in a significant worsening of motor deficits (Fig. 3B), and the tauSetdb1 allele conferred a strong trend (P < 0.07) towards further shortening of the survival time of these animals (Fig. 5B). These findings effectively rule out the hypothetical possibility that the artificial elevation of H3K9 methylation in our Setdb1 overexpressing mice was not sufficiently high in order to compensate for the loss of MeCP2. Of note, Setdb1 transgenes in mice expressing wildtype Mecp2 appear target a select set of genes and appear to exert a therapeutic, antidepressant-like effect on mood-related behaviors without adverse consequences on the animals health (Jiang et al., 2010). These findings, taken together, imply that brains of mice with normal levels of MeCP2 are able to tolerate the artificial hypermethylation of H3K9, but the same manipulation applied to the Mecp2 deficient brain then leads to additional dysfunction and further decompensation of the animals’ RTT phenotypes. Presently, it remains unknown whether or not the Setdb1 and Mecp2 proteins functionally interact or co-localize in neuronal chromatin remodeling complexes. Our finding that neuronal overexpression of Setdb1 is tolerated in an otherwise normal brain, but not in RTT, is of interest from the viewpoint of a related finding in Huntington’s disease, a neurodegenerative disorder caused by polyglutamine repeats which is associated with transcriptional dysregulation (Butler and Bates, 2006). Interestingly, both Setdb1 expression and levels of H3K9me3 are increased in Huntington’s striatum (Lee et al., 2008; Ryu et al., 2006; Stack et al., 2007). These findings are largely of a correlative nature and while further investigations are required, the evidence thus far would suggest dysregulation of Setdb1-mediated histone methylation worsens neuronal disease in the Huntington’s brain as it does in the Mecp2 mutant mice of the present study, while a similar level of Setdb1 activity in normal brain does not result in an overt phenotype.
To date, little is known about the molecular signaling pathways regulating Setdb1 expression and function in brain. A number of chromatin-associated proteins that interact with Setdb1, including mSin3B (Yang et al., 2003), histone deacetylases (HDAC) 1 and 2 (Ng and Bird, 2000), and tripartite motif protein 28 (Trim28), also known as KAP-1 co-repressor (Schultz et al., 2002), are expressed at moderate or high levels in adult brain (Lein et al., 2007). Furthermore, gene deletion studies suggest that the histone acetyl-transferase, CREB-binding protein (CBP), inhibits Setdb1 expression in striatal neurons by limiting Ets-2 transcription factor activity (Lee et al., 2008). However, the precise role of Setdb1 in developing and mature brain, including its potential contribution to neurological disease, requires further investigation. Notably, in the Setdb1 overexpressing mice of the present study, histone trimethylation at pericentric repeats was increased, which is in good agreement with a recent report linking CBP deficiency in brain to increased expression of Setdb1, which was accompanied by increased H3K9me3 and expansion of pericentric heterochromatin (Lee et al., 2008). Therefore, at least in brain, Setdb1 could be involved in the epigenetic regulation of pericentromeric satellites and other DNA repeats, in addition to its well described interaction with repressor and silencing complexes in euchromatin (Ayyanathan et al., 2003; Li et al., 2006; Lyst et al., 2006; Takada et al., 2007; Wang et al., 2007; Wang et al., 2003) which in mature neurons are likely to affect less than 1% of annotated genes (Jiang et al., 2010). Indeed, a role for Setdb1 in the regulation of DNA repeat chromatin is supported by the observation that this methyl-transferase is recruited by heterochromatin protein HP-1 to mediate histone methylation in repetitive DNA in various mammalian cell lines (Verschure et al., 2005), and at pericentromeric repeats in murine embryonic stem cells lacking Suv(3)9h1/2 histone methyl-transferase (HMTase) activity (Kourmouli et al., 2005). Given that Suv(3)9h1 interacts with MeCP2 (Lunyak et al., 2002), one could speculate that neuronal overexpression of this particular methyl-transferase—in contrast to the Setdb1 experiments presented here—could rescue the RTT phenotype of Mecp2 mutant mice. However, because H3K9 methylation levels in the pericentromeric satellite repeats of mature neurons lacking Mecp2 did not differ from wildtype controls, a more likely scenario is that neither SuV(3)9h1/2 nor Setdb1 or other H3K9-specific methyl-transferase transgenes prevent or ameliorate neuronal disease resulting from the loss of Mecp2.
However, it is likely that the Setdb1 transgenes of the present study affected histone H3K9 methylation, in addition to pericentric heterochromatin and other repeats, also at several single copy genes. The small subset of annotated genes targeted by Setdb1 in mature neurons includes the N-methyl-D-aspartate (NMDA) receptor subunit Grin2b and additional glutamate receptor genes (Jiang et al., 2010). Therefore, it remains possible that Setdb1-mediated histone H3K9 hypermethylation and repressive chromatin remodeling could result in decreased excitatory neurotransmission in the Mecp2 deficient mice of the present study, thereby further potentiating the neuronal signaling defects in the animals (Dani et al., 2005; Nelson et al., 2008).
Of note, there is evidence that in some brain regions, including the hypothalamus, Mecp2 both positively and negatively regulates a substantial number of mRNAs, suggesting that this protein may play a more complex role in transcriptional regulation than repression alone (Chahrour et al., 2008). Furthermore, a recent case diagnosed with the congenital variant of RTT was linked to a truncating mutation in FOXG1 (Ariani et al., 2008), which encodes a brain-specific transcriptional factor that interacts with JARID1B (Roesch et al., 2008), a histone demethylase with specificity for the H3K4 residue (Xiang et al., 2007; Yamane et al., 2007). H3K4 methylation is commonly associated with open chromatin and active gene expression (Shilatifard, 2008). Thus, it remains possible that artificial manipulation of histone methylation marks other than H3K9 could profoundly modify the RTT phenotype of Mecp2 mutant mice.
The authors would like to thank Dr. Steven Jones and the team of the Transgenic Animal Modeling Core, and Dr. Roger Davis and staff of the Program in Molecular Medicine, University of Massachusetts Medical School. We also thank Andrea Allersdorfer for technical assistance and Caroline Connor for helpful comments on the manuscript. Supported by grants from the National Institutes of Health and the International Rett Syndrome Foundation.
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